# Patent

## Title
Transgenic reporter system that reveals expression profiles and regulation mechanisms of alternative splicing in mammalian organisms

## Abstract

An object of the present invention is to develop a new alternative splicing reporter system and to provide a method for detecting alternative splicing patterns in a mammalian multicellular organism more precisely, a method for identifying efficiently substances and gene regions that affect alternative splicing in a mammalian multicellular organism, and the like by utilizing the alternative splicing reporter system. Specifically, the present invention relates to a method for detecting alternative splicing in a mammalian multicellular organism, and a method for identifying substances and gene regions that affect alternative splicing in a mammalian multicellular organism, which use a DNA construct in which at least two different reporter genes are inserted into a specific gene that undergoes alternative splicing, or a combination of DNA constructs (a combination of at least two different DNA constructs) in which DNA construct a reporter gene is inserted into a specific gene that undergoes alternative splicing.

## Background
TECHNICAL FIELD
The present invention relates to a method for detecting alternative splicing in a mammalian multicellular organism, a method for identifying substances and gene regions that affect alternative splicing in a mammalian multicellular organism, and the like.
PRIORITY
The present application is a U.S. National Phase of PCT/JP2011/003059, filed May 31, 2011, which claims the benefit of U.S. Application No. 61/350,420, filed on Jun. 1, 2010, the entire contents of which are incorporated by reference herein in their entirety.
REFERENCE TO SEQUENCE LISTING
This application includes a Sequence Listing as a text file named “87331-023710US-858742 SEQ LIST.txt” created Nov. 28, 2012, and containing 9,581 bytes. The material contained in this text file is incorporated by reference in its entirety for all purposes.
BACKGROUND ART
Genome projects have shown that metazoans generate a hugely diverse proteome from a limited number of genes. This finding underscores the importance of alternative splicing, through which a single gene can generate multiple structurally and functionally distinct protein isoforms. Moreover, recent transcriptome analyses with splicing-sensitive microarrays or deep sequencers have revealed that alternative splicing occurs in more than 90% of multi-exon genes in human (NPL 1) and over 60% of these cases are regulated in a tissue- and cell type-specific manner (NPL 2). Alternative splicing is regulated by auxiliary cis-elements with regulatory proteins that enhance or repress splicing of adjacent exons (NPL 3, 4) however, the mechanism by which a number of genes are regulated in various tissue-specific manner by a limited number of regulatory factors remains unclear.
In mammals, fibroblast growth factor-receptor 2 (FGFR2) is one of the best characterized gene in which mutually exclusive alternative splicing produces two isoforms. Exon 8 (also termed IIIb) isoform is specifically expressed in epithelial tissues, whereas exon 9 (or IIIc) isoform is selected in non-epithelial or mesenchymal tissues (NPL 5, 6). The structural difference between two splice isoforms markedly affects the specificity of ligand-receptor binding (NPL 7, 8, 9), and exon switching is shown to be essential for development in the mouse (NPL 10, 11). Several factors have been identified which positively or negatively regulate either of alternative exons of FGFR2 independently. For exon 8 regulation, Del Gatto-Konczak et al. found that heterogeneous nuclear ribonucleoprotein, hnRNP A1, binds to exon 8 (also termed K-SAM exon) as ESS (exonic splicing silencer) and represses its inclusion (NPL 12). Carstens et al. found the polypyrimidine tract binding protein (PTB) represses exon 8 inclusion through ISS-1 and ISS-2 (intronic splicing silencers-1 and 2) (NPL 13). Warzecha et al. recently cloned RBM35a and RBM35b as epithelia-specific activators of exon 8 inclusion, and renamed them epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESPR2), respectively (NPL 14). For exon 9 regualtion, Chen et al. found that Tra2beta represses the selection of exon 9 (NPL 15). Baraniak et al. reported that Fox2 represses selection of exon 9 through binding to a UGCAUG sequence in intron 8 (NPL 16). Hovhannisyan et al. found that a hnRNP M binds to ISS-3 and represses inclusion of exon 9 (NPL 17). Mauger et al. showed that hnRNP H and F interact with Fox2 and repress exon 9 inclusion (NPL 18). Also, presence of unknown enhancer is speculated for exon 9 inclusion through ISE (intronic splicing enhancer) in intron 9 (NPL 14).
CITATION LIST
Non Patent Literature


    NPL 1: Pan Q, Shai O, Lee L J, Frey B J, Blencowe B J (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet. 40: 1413-1415.
    NPL 2: Wang E T, Sandberg R, Luo S, Khrebtukova I, Zhang L, et al. (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456: 470-476.
    NPL 3: Matlin A J, Clark F, Smith C W (2005) Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol 6: 386-398.
    NPL 4: Black D L (2003) Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72: 291-336.
    NPL 5: Gilbert E, Del Gatto F, Champion-Arnaud P, Gesnel M C, Breathnach R (1993) Control of BEK and K-SAM splice sites in alternative splicing of the fibroblast growth factor receptor 2 pre-mRNA. Mol Cell Biol 13: 5461-5468.
    NPL 6: Savagner P, Valles A M, Jouanneau J, Yamada K M, Thiery J P (1994) Alternative splicing in fibroblast growth factor receptor 2 is associated with induced epithelial-mesenchymal transition in rat bladder carcinoma cells. Mol Biol Cell 5: 851-862.
    NPL 7: Ornitz D M, Xu J, Colvin J S, McEwen D G, MacArthur C A, et al. (1996) Receptor specificity of the fibroblast growth factor family. J Biol Chem 271: 15292-15297.
    NPL 8: Eswarakumar V P, Lax I, Schlessinger J (2005) Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 16: 139-149.
    NPL 9: Zhang X, Ibrahimi O A, Olsen S K, Umemori H, Mohammadi M, et al. (2006) Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem 281: 15694-15700.
    NPL 10: De Moerlooze L, Spencer-Dene B, Revest J M, Hajihosseini M, Rosewell I, et al. (2000) An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signalling during mouse organogenesis. Development 127: 483-492.
    NPL 11: Eswarakumar V P, Monsonego-Ornan E, Pines M, Antonopoulou I, Morriss-Kay G M, et al. (2002) The IIIc alternative of Fgfr2 is a positive regulator of bone formation. Development 129: 3783-3793.
    NPL 12: Del Gatto-Konczak F, Olive M, Gesnel M C, Breathnach R (1999) hnRNP A1 recruited to an exon in vivo can function as an exon splicing silencer. Mol Cell Biol 19: 251-260.
    NPL 13: Carstens R P, Wagner E J, Garcia-Blanco M A (2000) An intronic splicing silencer causes skipping of the IIIb exon of fibroblast growth factor receptor 2 through involvement of polypyrimidine tract binding protein. Mol Cell Biol 20: 7388-7400.
    NPL 14: Warzecha C C, Sato T K, Nabet B, Hogenesch J B, Carstens R P (2009) ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing. Mol Cell 33: 591-601.
    NPL 15: Chen X, Huang J, Li J, Han Y, Wu K, et al. (2004) Tra2betal regulates P19 neuronal differentiation and the splicing of FGF-2R and GluR-B minigenes. Cell Biol Int 28: 791-799.
    NPL 16: Baraniak A P, Chen J R, Garcia-Blanco M A (2006) Fox-2 mediates epithelial cell-specific fibroblast growth factor receptor 2 exon choice. Mol Cell Biol 26: 1209-1222.
    NPL 17: Hovhannisyan R H, Carstens R P (2007) Heterogeneous ribonucleoprotein m is a splicing regulatory protein that can enhance or silence splicing of alternatively spliced exons. J Biol Chem 282: 36265-36274.
    NPL 18: Mauger D M, Lin C, Garcia-Blanco M A (2008) hnRNP H and hnRNP F complex with Fox2 to silence fibroblast growth factor receptor 2 exon IIIc. Mol Cell Biol 28: 5403-5419.


SUMMARY OF INVENTION
Technical Problem
An object of the present invention is to develop a new alternative splicing reporter system in a mammalian organism and to provide a method for detecting alternative splicing patterns in a mammalian multicellular organism more precisely, a method for identifying efficiently substances and gene regions that affect alternative splicing in a mammalian multicellular organism, and the like by utilizing the alternative splicing reporter system.
Solution to Problem
Since alternative splicing of pre-mRNAs is essential for generating tissue-specific diversity in proteome, elucidating its regulatory mechanism is indispensable to understand developmental process or tissue-specific functions. The present inventors have investigated tissue-specific regulation of mutually exclusive selection of alternative exons using transgenic splicing reporters in nematode, because the present inventors hypothesized that this implies the typical molecular mechanism of alternative splicing. So far, mutually exclusive splicing regulation has been explained by the outcome from the balance of multiple regulators that enhance or repress either of alternative exons discretely. However, this “balance” model is open to questions how to ensure the selection of only one appropriate exon out of several candidates and how to switch them. To answer these questions, the present inventors generated an original bichromatic fluorescent splicing reporter system for mammals using fibroblast growth factor-receptor 2 (FGFR2) gene as model. By using this splicing reporter, the present inventors demonstrated that FGFR2 gene is regulated by the “switch-like” mechanism”, in which key regulators modify the ordered splice-site recognition of two mutually exclusive exons, eventually ensure single exon selection and their distinct switching. Also this finding elucidated the evolutionally conserved “splice code”, in which combination of tissue-specific and broadly expressed RNA binding proteins regulate alternative splicing of specific gene in tissue-specific manner. These findings provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand developmental process or tissue-specific functions.
The present invention is based on the above-mentioned experiences or discoveries by the present inventors, and means for solving the above-mentioned problems are as follows. Specifically,
A first method for detecting an alternative splicing of a specific gene in a mammalian multicellular organism of the present invention includes:

    
    
        (a) introducing into the mammalian multicellular organism a DNA construct such that at least two different reporter genes are inserted into the specific gene which undergoes the alternative splicing, wherein the reporter genes are inserted so that transcripts of the different reporter genes are each fused with each of at least two different mature mRNAs generated by alternative splicing; and
        (b) detecting the alternative splicing of the specific gene in the mammalian multicellular organism by the expression of the reporter genes.
    
    


A first method for testing whether or not a compound to be tested affects an alternative splicing of a specific gene of the present invention includes:
(a) introducing into a mammalian multicellular organism a DNA construct such that at least two different reporter genes are inserted into the specific gene which undergoes the alternative splicing, wherein the reporter genes are inserted so that transcripts of the different reporter genes are each fused with each of at least two different mature mRNAs generated by the alternative splicing;

(b) allowing the mammalian multicellular organism to contact the compound to be tested;

(c) detecting the alternative splicing of the specific gene in the mammalian multicellular organism by the expression of the reporter genes; and

(d) determining whether or not the expression of the reporter genes detected in the (c) has changed compared to the expression of the reporter genes in a control which is not allowed to contact the compound to be tested.

A first method for identifying a gene region affecting an alternative splicing of a specific gene of the present invention includes:

    
    
        (a) introducing into a mammalian multicellular organism a DNA construct such that at least two different reporter genes are inserted into the specific gene which undergoes the alternative splicing,
        wherein the reporter genes are inserted so that transcripts of the different reporter genes are each fused with each of at least two different mature mRNAs generated by the alternative splicing;
        (b) treating the mammalian multicellular organism with a mutagen;
        (c) detecting the alternative splicing of the specific gene in the mammalian multicellular organism by the expression of the reporter genes;
        (d) selecting an individual in which the expression of the reporter genes detected in the (c) has changed compared to the expression of the reporter genes in a control which is not subjected to the mutagen treatment; and
        (e) identifying a mutated gene region in the individual.
    
    


A first method for identifying a region in a specific gene affecting an alternative splicing of the specific gene of the present invention includes:

    
    
        (a) introducing into a mammalian multicellular organism a DNA construct such that at least two different reporter genes are inserted into the specific gene into which a mutation has been introduced and which undergoes the alternative splicing,
        wherein the reporter genes are inserted so that transcripts of the different reporter genes are each fused with each of at least two different mature mRNAs generated by the alternative splicing;
        (b) detecting the alternative splicing of the specific gene in the mammalian multicellular organism by the expression of the reporter genes; and
        (c) determining whether or not the expression of the reporter genes detected in the (b) has changed compared to the expression of the reporter genes in a control in which a mutation has not been introduced into the specific gene.
    
    


A second method for detecting an alternative splicing of a specific gene in a mammalian multicellular organism of the present invention includes:

    
    
        (a) introducing into the mammalian multicellular organism a combination of DNA constructs, wherein in the DNA construct, a reporter gene is inserted into the specific gene which undergoes the alternative splicing, and the combination of DNA constructs satisfies all of the following conditions (i) to (iv):
        (i) reporter genes inserted into each DNA construct are different from each other,
        (ii) in each DNA construct, the reporter gene is inserted into the specific gene so that a transcript of the reporter gene is fused with a plurality of mature mRNAs generated by the alternative splicing,
        (iii) a transcript portion of the reporter gene in only one mature mRNA of the plurality of mature mRNAs generated by the alternative splicing from each DNA construct is translated in a correct reading frame, and
        (iv) only when a specific splicing is selected among alternative splicing patterns, the translation in a correct reading frame is induced in each DNA construct, and the specific splicing which induces the translation in a correct reading frame is different depending on each DNA construct; and
        (b) detecting the alternative splicing of the specific gene in the mammalian multicellular organism by the expression of the reporter genes.
    
    


A second method for testing whether or not a compound to be tested affects an alternative splicing of a specific gene of the present invention includes:

    
    
        (a) introducing into a mammalian multicellular organism a combination of DNA constructs,
        wherein in the DNA construct, a reporter gene is inserted into the specific gene which undergoes the alternative splicing, and the combination of DNA constructs satisfies all of the following conditions (i) to (iv):
        (i) reporter genes inserted into each DNA construct are different from each other,
        (ii) in each DNA construct, the reporter gene is inserted into the specific gene so that a transcript of the reporter gene is fused with a plurality of mature mRNAs generated by the alternative splicing,
        (iii) a transcript portion of the reporter gene in only one mature mRNA of the plurality of mature mRNAs generated by the alternative splicing from each DNA construct is translated in a correct reading frame, and
        (iv) only when a specific splicing is selected among alternative splicing patterns, the translation in a correct reading frame is induced in each DNA construct, and the specific splicing which induces the translation in a correct reading frame is different depending on each DNA construct;
        (b) allowing the mammalian multicellular organism to contact the compound to be tested;
        (c) detecting the alternative splicing of the specific gene in the mammalian multicellular organism by the expression of the reporter genes; and
        (d) determining whether or not the expression of the reporter genes detected in the (c) has changed compared to the expression of the reporter genes in a control which is not allowed to contact the compound to be tested.
    
    


A second method for identifying a gene region affecting an alternative splicing of a specific gene of the present invention includes:

    
    
        (a) introducing into a mammalian multicellular organism a combination of DNA constructs,
        wherein in the DNA construct, a reporter gene is inserted into the specific gene which undergoes the alternative splicing, and the combination of DNA constructs satisfies all of the following conditions (i) to (iv):
        (i) reporter genes inserted into each DNA construct are different from each other,
        (ii) in each DNA construct, the reporter gene is inserted into the specific gene so that a transcript of the reporter gene is fused with a plurality of mature mRNAs generated by the alternative splicing,
        (iii) a transcript portion of the reporter gene in only one mature mRNA of the plurality of mature mRNAs generated by the alternative splicing from each DNA construct is translated in a correct reading frame, and
        (iv) only when a specific splicing is selected among alternative splicing patterns, the translation in a correct reading frame is induced in each DNA construct, and the specific splicing which induces the translation in a correct reading frame is different depending on each DNA construct;
        (b) treating the mammalian multicellular organism with a mutagen;
        (c) detecting the alternative splicing of the specific gene in the mammalian multicellular organism by the expression of the reporter genes;
        (d) selecting an individual in which the expression of the reporter genes detected in the (c) has changed compared to the expression of the reporter genes in a control which is not subjected to the mutagen treatment; and
        (e) identifying a mutated gene region in the individual.
    
    


A second method for identifying a region in a specific gene affecting an alternative splicing of the specific gene of the present invention includes:

    
    
        (a) introducing into a mammalian multicellular organism a combination of DNA constructs,
        wherein in the DNA construct, a reporter gene is inserted into the specific gene into which a mutation has been introduced and which undergoes the alternative splicing, and the combination of DNA constructs satisfies all of the following conditions (i) to (iv):
        (i) reporter genes inserted into each DNA construct are different from each other,
        (ii) in each DNA construct, the reporter gene is inserted into the specific gene so that a transcript of the reporter gene is fused with a plurality of mature mRNAs generated by the alternative splicing,
        (iii) a transcript portion of the reporter gene in only one mature mRNA of the plurality of mature mRNAs generated by the alternative splicing from each DNA construct is translated in a correct reading frame, and
        (iv) when a specific splicing is selected among alternative splicing patterns, the translation in a correct reading frame is induced in each DNA construct, and the specific splicing which induces the translation in a correct reading frame is different depending on each DNA construct;
        (b) detecting the alternative splicing of the specific gene in the mammalian multicellular organism by the expression of the reporter genes; and
        (c) determining whether or not the expression of the reporter genes detected in the (b) has changed compared to the expression of the reporter genes in a control in which a mutation has not been introduced into the specific gene.
    
    


The present invention can solve conventional problems, can attain the above-mentioned object, and can provide a method for detecting alternative splicing patterns in a mammalian multicellular organism, a method for identifying efficiently substances and gene regions that affect alternative splicing in a mammalian multicellular organism, and the like.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows single cell detection of splicing events with fluorescent splicing reporters. Overview: Construction of fluorescent alternative splicing reporter mini-genes.
FIGS. 2A-2D show schematic structure of fluorescent reporter mini-genes and expected mRNAs. (A, B) Two-construct fluorescent alternative splicing Cloning reporter mini-genes for mutually exclusive exons (A) and a cassette exon (B). (C, D) Single-construct fluorescent alternative splicing Cloning reporter mini-genes for a cassette exon (C) and mutually exclusive exons (D). Boxes indicate exons. CE, cassette exon. Alternative exons to be analyzed are in black. GFP, RFP and GST cDNAs are indicated in gray. Open circles, diamonds and double arrowheads indicate artificial translation initiation codons, termination codons and artificial frame-shifts, respectively. attB sites are indicated with arrows. Open reading frames of expected mRNAs are colored.
FIGS. 3A-3B show construction of an ‘Expression’ clone by ‘2-fragment’ recombination reaction utilizing MULTISITE GATEWAY® vector construction system. (A) Cloning DNA fragments of interest in ‘Entry’ vectors by ‘BP’ reaction. attB-flanked PCR products and two MULTISITE GATEWAY® ‘Donor’ vectors are used in separate ‘BP’ recombination reactions to generate two ‘Entry’ clones, one with attL1 and attR5 sites, and the other with attL5 and attL2 sites. att sites are not palindromic and have an orientation. The direction of the arrowhead designates the orientation of each att site in relation to the insert; the attB5 or attP5 site is designated with “r” when the arrowhead does not point towards the insert. [It appears as attBSr or attPSr in the left panel.] (B) Construction of an ‘Expression’ clone by ‘LR’ reaction. The two ‘Entry’ clones and a ‘Destination’ vector are used together in ‘ LR’ re-combination reaction to create an ‘Expression’ clone containing the two DNA fragments.
FIGS. 4A-4F show examples of the analysis. (A, B) Visualization of mutually exclusive alternative splicing in C. elegans. (A) Schematic structure of a trio of reporter mini-genes to monitor selection profiles of three mutually exclusive exons (a, b and c). The promoter, the constitutive first intron and the 3′ cassette were provided by a ‘Destination’ vector. Two termination codons (diamonds) are introduced into two of the alternative exons in each construct. attB sites are indicated with arrows. (B) A microphotograph of a fluorescent alternative splicing reporter worm carrying the mini-genes in (A). Expression of Venus (green), mRFP (red) and ECFP (blue) shows tissue-specificity. (C, D) Visualization of virus-induced intron retention. (C) Schematic structure of a reporter mini-gene to monitor splicing of an alternatively retained intron, and mRNAs derived from it. RFP protein is produced only when the alternative intron is properly spliced. (D) Microphotographs of uninfected (left) and Venus-HSV-2-infected (right) HeLa cells. All uninfected cells express RFP (magenta), while cells infected with Venus-HSV-2 (green) shut off RFP expression. (E, F) Visualization of tissue-specific alternative splicing in mice. (E) Schematic structure of a reporter mini-gene to monitor selection of mutually exclusive alternative exons, and mRNAs derived from it. GFP protein is produced when exon a alone is selected and RFP is produced when exon b alone is selected. (F) Microphotographs of a mouse embryo at E14.5. (Left) Expression of GFP is detected in epidermis (arrowheads). (Right) Expression of RFP is detected in the nervous system and mesenchymal tissues.
FIG. 5 shows the meaning of each symbol used in the FIGS. 1 to 4.
FIGS. 6A-6D show the construction of FGFR2 splicing reporter vector and their expression patterns. (A) Scheme of FGFR2 splicing reporter vector. The genomic fragment of mouse FGFR2 including exon 7 through 10 was amplified and introduced into the reporter vector containing a CAGGS promoter and RFP-EGFP with different reading frames. Modified glutathione-S-transferase gene (indicated as “G”) was inserted in front of the exon 7 in-frame. A schematic representation of the mRNA derived from the reporter under the alternative splicing regulation is also shown; the numbers indicate the reading frames. (B) Expression pattern of splicing reporter in vitro. The reporter vector was introduced into two rat prostate cancer cell lines AT-3 and DT-3, which have different cell-type specificities. Scale=200 micrometer. (C) Expression pattern of splicing reporter in vivo. Fluorescence images of transgenic reporter mouse embryos at E14.5. Tg(+) is an embryo carrying the reporter vector, and Tg(−) is one of its litter-mate lacking the vector. Arrowheads in Tg(+) indicate EGFP signals with the patterns of whiskers (upper arrowhead) and the edge of a limb (lower arrowhead), both of which are magnified and indicated by white rectangles in the upper left-hand and lower right-hand corners, respectively (scale=1 mm). (D) Sections from transgenic reporter mouse embryos at E16.5. Each panel shows sections from the indicated tissues, the upper one from Tg(+) and the lower from Tg(−). Portions expressing the EGFP signal are indicated by white arrows (scale=100 micrometer).
FIGS. 7A-7B show the unbalanced sequence of 3′ splice sites is essential for mutually exclusive exon selection. (A) Scheme for 3′ splice site mutation on exons 8 and 9. The sequence of the weak 3′ splice site of exon 8 is TGTTCTAGca (SEQ ID NO: 40). The sequence of the strong 3′ splice site of exon 9 is TTTTCTAGgc (SEQ ID NO: 41). Uppercase letter is intron and lowercase is exon sequence. White characters on a black background indicate mismatches from the conserved consensus sequence in the 3′ splice site and poly-pyrimidine moiety, and underline indicate mutated sequence. The gray arrows with “primer” represent the positions amplified in RT-PCR. (B) RT-PCR from AT-3 and DT-3 cells trans fected the indicated vectors. Splice products were digested with EcoRV, which uniquely cuts the PCR product containing exon 9. Each band was identified and indicated with the scheme of splice products. Arrowheads indicate nonspecific PCR products, which was confirmed by sequencing. The asterisk indicates the splice product came from double inclusion of exon 8 and exon 9. The bar graph shows the amount of each splicing product, and is based on calculations from three independent experiments; the mean value for each splice product is show in the respective column with an error bar showing the SD (standard error).
FIGS. 8A-8C show the promoted selection of exon 8 by disruption of 3′ splice site of exon 9. (A) Scheme of 3′ and 5′ splice site mutation on exon 9. Uppercase letter is intron and lowercase is exon sequence, white characters on a black background indicates mutated sequence. (B) RT-PCR from AT-3 cells into which the indicated vectors were introduced. Arrowshead indicate aberrant spliced product that used the 5′ cryptic splice site inside exon 9. The bar graph, which represents the amount of each splicing product, is based on calculations from three independent experiments; the mean value for each splice product is show in the respective column with an error bar showing the SD (standard error). (C) Results of the in vitro splice site recognition assay. The scheme for exon 9 shows the position of the splice site mutation as “x”. “X-link” shows the presence or absence of UV-induced crosslinks in samples after the in vitro splicing reaction. “U1 oligo” and “U2 oligo” represent the digestion of RNA samples by RNaseH with complementary oligos for U1 or U2. The band shown by arrowheads with asterisk may be a probe crosslinked with U1 that binds to the cryptic 5′ splice site inside exon 9, because it was detected in the 5′ ss mutated probe and digested with U1 oligo.
FIGS. 9A-9C show the identification of silencing elements for exon 9 recognition. (A) Scheme of cis-mutation experiment on UGCAUG and ISE/ISS-3 which is located upstream of exon 9. UGCAUGCAUG (SEQ ID NO: 42) was substituted for UACGUACGUG (SEQ ID NO: 43) to disrupt the binding to the RNA-binding protein of Fox. White characters on a black background indicated mutated sequences or deletions. (B) RT-PCR from AT-3 cell into which the indicated vectors were introduced. The bar graph shows the amount of each splicing product, and is based on calculations from three independent experiments; the mean value for each splice product is show in the respective column, with an error bar showing the SD (standard error). (C) RT-PCR from AT-3 and DT-3 cells showing amplified endogenous FGFR2, Fox1, Fox2, ESRPI, and ESRP2. The arrowhead in ESRPI corresponds to two splice isoforms which was confirmed by sequencing.
FIGS. 10A-10C show the Foxs and ESRPs which promote switching to exon 8 through repressing exon 9. (A) RT-PCR from HeLa cell transfected the wild-type reporter and indicated cDNA expression vectors with or without Fox2 siRNA. The bar graph shows the amount of each splicing product. (B) Fluorescent microscopy image of HeLa cell transfected the wild-type reporter with indicated cDNA expression vectors (scale bar=200 micrometer). (C) Result of an in vitro splice site recognition assay. The scheme for exon 9 RNA probe shows the position of UGCAUG and ISE/ISS-3. The asterisk shows the probe crosslinked with U1, which binds to the cryptic 5′ splice site inside exon 9.
FIG. 11 shows the expression pattern of Foxs and ESRPs in splicing reporter mouse embryos. Sections from transgenic reporter embryos at E16.5. EGFP signal showed the exon 8 splicing pattern, and in situ hybridization was performed with indicated probes using serial sections. The EGFP signal is indicated by a white arrow. The violet signal, indicating mRNA localization, is shown by arrows, and nuclei were counterstained with Methyl Green (scale bar=100 micrometer).
FIGS. 12A-12B show the model for tissue-specific splicing regulation of FGFR2 gene. (A) “The Knight's Fork” regulation model: In non-epithelial or mesenchymal tissues, exon 9 is chosen as “primary” exon due to its stronger 3′ splice site than exon 8 (“default” selection to choose “primary” exon). In epithelial tissues, “key regulators” repress exon 9 utilizing its 3′ splice site dependency for exon recognition and cause switch to “secondary” exons (“alternative” selection to choose “secondary” exon). A small number of “key regulators” can control two mutually exclusive exons through modifying ordered splice-site recognitions in a tissue-specific manner, resembling the way that a chess piece can simultaneously attack a rook and check the king. (B) In nematode, mutually exclusive splicing of the worm FGFR homologue gene of egl-15 is regulated by the cooperation of broadly expressed Fox-1/ASD-1 family and the muscle-specific RNA binding motif protein (RBMs) of SUP-12 (a worm homologue of mRBM24), which act together to repress inclusion of alternative exon 5B to promote muscle-specific expression of exon 5A. In the case of mammalian FGFR2, the Fox family cooperates with the tissue-specific factor ESRPs (RBM35a and b) to repress alternative inclusion of exon 9 and to promote epithelial tissue-specific expression of exon 8.
FIG. 13 shows Fox and ESRP which promote exon switching through repressing exon 9 via UGCAUG and ISE/ISS-3. RT-PCR from HeLa cell transfected the indicated wild-type or cis-mutated reporter vectors and Fox2 and/or ESRP1 expression vectors with or without Fox2 siRNA. When Fox2 was transfected with the UGCAUG site-mutated reporter, the promoting effect of Fox2 on exon 8 inclusion was lost (FIG. 13, lane 7). Overexpression of ESRP1 still promoted exon switching of the UGCAUG-mutated reporter, but the ratio of exon 8 selection was slightly reduced (FIG. 13, lane 8) in comparison with the wild-type reporter (FIG. 13, lane 3). To the contrary, when ISE/ISS-3 was mutated, the promotion effect of ESRP on the exon switching was significantly reduced (FIG. 13, lanes 13), and the effect of Fox2 remained (FIG. 13, lanes 12 and 15). When both UGCAUG and ISE/ISS-3 were mutated, neither Fox2 nor ESRP1 caused a drastic switching any more (FIG. 13, lane 16-20). The bar graph shows the amount of each splicing product, and is based on calculations from three independent experiments; the mean value for each splice product is show in the respective column, with an error bar showing the SD (standard error).
FIG. 14 shows a strategy for screening the compound which can operate alternative splicing.



DESCRIPTION OF EMBODIMENTS
First and Second Alternative Splicing Reporter Systems
In a first aspect, the present invention relates to an alternative splicing reporter system in a mammalian multicellular organism using a DNA construct in which at least two different reporter genes are inserted into a specific gene that undergoes alternative splicing (for example, see FIGS. 2C and 2D). In a second aspect, the present invention relates to an alternative splicing reporter system in a mammalian multicellular organism using a combination of DNA constructs (a combination of at least two different DNA constructs) in which DNA construct a reporter gene is inserted into a specific gene that undergoes alternative splicing (for example, see FIGS. 2A and 2B).
Both of the first and second systems allows the detection of alternative splicing patterns of a specific gene in vivo (or in vitro, or ex vivo) in mammalian multicellular organisms by introducing a DNA construct (or combination of DNA constructs), into which at least two different reporter genes are inserted in total, into a mammalian multicellular organism. The first and second systems each can be suitably utilized for “(1) a method for detecting alternative splicing of a specific gene in a mammalian multicellular organism”, “(2) a method for testing whether or not a compound to be tested affects alternative splicing of a specific gene”, “(3) a method for identifying a gene region affecting alternative splicing of a specific gene”, and “(4) a method for identifying a region in a specific gene affecting alternative splicing of the specific gene” of the present invention, which will be described later.
It was difficult to precisely predict tissue-specific and/or stage-specific alternative splicing patterns in vivo from the results of conventional in vitro studies on alternative splicing using cultured cells, etc. Use of the first and second systems enables direct and accurate detection of tissue-specific and/or stage-specific alternative splicing patterns in vivo in mammalian multicellular organisms. Thus, it is considered that the first and second systems can become powerful experimental tools for elucidating mechanisms of alternative splicing in mammalian multicellular organisms.
<Multicellular Organism>
In the first and second systems, the “mammalian multicellular organism” is not particularly limited and can be appropriately selected depending on the application as long as it consists of many cells and has mechanisms of alternative splicing, including, for example, mouse, and rat. Namely, the mammalian used in the present invention is preferably non-human animal such as rodent animals (i.e. mouse, rat, etc.). The “mammalian multicellular organism” of the present invention includes the cell derived from mammalian animal. Namely, present invention encompasses the method using the cells derived from mammalian animals.
<Specific Gene Undergoing Alternative Splicing>
The “specific gene that undergoes alternative splicing” (hereinafter, may be simply referred to as “specific gene”) in the first and second systems is not particularly limited and can be appropriately selected depending on the application as long as it is a gene of mammalian multicellular organisms and can produce multiple isoforms of mature mRNAs consisting of different combinations of exons as a result of alternative splicing.
Here, the “alternative splicing” refers to a phenomenon in which multiple isoforms of mature mRNAs consisting of different combinations of exons are produced in cells by varying patterns of splicing when introns are removed from pre-mRNAs by RNA splicing. This phenomenon enables organisms having alternative splicing machinery to produce different proteins from a single gene.
<Reporter Gene>
In the first and second systems, the “reporter gene” is not particularly limited and can be appropriately selected depending on the application. Examples of the reporter gene include fluorescent protein genes such as a green fluorescent protein (GFP) and a red fluorescent protein (RFP), enzyme genes that catalyze chromogenic reaction or color-developing reaction, or luminous reaction, and the like. In the first and second systems, at least two different reporter genes are used in total. The combination thereof is not particularly limited and can be appropriately selected depending on the application as long as their expressions can be distinguished from each other based on the difference of fluorescence or developed color. For example, a combination of a green fluorescent protein (GFP) and a red fluorescent protein (RFP), and the like can be suitably used.
<DNA Construct>
—DNA Construct of First System—
The DNA construct in the first system is one in which at least two different reporter genes are inserted into a specific gene that undergoes alternative splicing. The at least two different reporter genes are inserted into the specific gene such that transcripts of the at least two different reporter genes are each fused with each of at least two different mature mRNAs generated by alternative splicing of the specific gene.
Preferably, the at least two different reporter genes are each connected to a different exon which is included in at least two different transcripts (mature mRNAs) generated by alternative splicing of the specific gene (for example, see FIGS. 2C and 2D). Each reporter gene may be connected upstream (5′ side) of each of the exons, or may be connected downstream (3′ side) thereof; however, each reporter gene is required to be connected to each of the exons such that each reporter gene is translated in a correct reading frame only when each exon, to which each reporter gene is connected, is translated in a correct reading frame. Such structure of the DNA construct allows to check whether each exon, to which each reporter gene is connected, has been translated in a correct reading frame when the expression of each reporter gene is detected. That is, analysis of respective expression patterns of the at least two different reporter genes makes it possible to check alternative splicing patterns of the specific gene comprehensively.
—DNA Construct of Second System—
On the other hand, the DNA construct in the second system is one in which a reporter gene is inserted into a specific gene that undergoes alternative splicing. The second system is characterized in that a combination of such DNA constructs (combination of at least two different DNA constructs) is used. Here, the combination of DNA constructs satisfies all conditions of the following (i) to (iv):
(i) reporter genes inserted into each DNA construct are different from each other;
(ii) in each DNA construct, the reporter gene is inserted into the specific gene so that a transcript of the reporter gene is fused with a plurality of mature mRNAs generated by the alternative splicing;
(iii) a transcript portion of the reporter gene in only one mature mRNA of the plurality of mature mRNAs generated by the alternative splicing from each DNA construct is translated in a correct reading frame; and
(iv) only when a specific splicing is selected among alternative splicing patterns, the translation in a correct reading frame is induced in each DNA construct, and the specific splicing which induces the translation in a correct reading frame is different depending on each DNA construct.
Preferably, each reporter gene in each DNA construct is connected to one of different exons which are included in at least two different transcripts (mature mRNAs) generated by alternative splicing of the specific gene (for example, see FIGS. 2A and 2B). Each reporter gene may be connected upstream (5′ side) of each of the exons, or may be connected downstream (3′ side) thereof; however, each reporter gene is required to be connected to each of the exons such that each reporter gene is translated in a correct reading frame only when each exon, to which each reporter gene is connected, is translated in a correct reading frame. Use of combination of DNA constructs constructed in such a way allows to check whether each exon, to which each reporter gene is connected, has been translated in a correct reading frame when the expression of each reporter gene is detected. That is, analysis of each expression pattern of each reporter gene in the combination of DNA constructs makes it possible to check alternative splicing patterns of the specific gene comprehensively.
In the DNA constructs of the first and second systems, the specific gene and the reporter gene are preferably linked downstream of a promoter so that they can be expressed in a mammalian multicellular organisms. The promoter is not particularly limited and can be appropriately selected depending on the application. Examples thereof include tissue-specific promoters such as the CAGGS promoter.
The DNA construct can be constructed by any method without limitation, and known techniques can be appropriately utilized. For example, the DNA construct can be constructed by integrating a reporter cassette, in which cDNA of the reporter gene is inserted into the genomic DNA fragment of the specific gene, into an expression vector that includes a desired promoter.
For example, DNA construct used for the present invention can be made by the method shown in FIG. 1 or 3.
Hereinafter, various methods according to the present invention utilizing the first and second alternative splicing reporter systems will be described.
((1) Method for Detecting Alternative Splicing of a Specific Gene in a Mammalian Multicellular Organism)
The method for detecting alternative splicing of a specific gene in a mammalian multicellular organism of the present invention comprises the steps of (a) introducing into the mammalian multicellular organism a DNA construct in the first system or a combination of DNA constructs in the second system (each contain the specific gene and the reporter gene), and (b) detecting the alternative splicing of the specific gene in the mammalian multicellular organism by detecting the expression of the reporter genes.
<Step (a)>
In the step (a), a DNA construct in the first system or a combination of DNA constructs in the second system is introduced into the mammalian multicellular organism to generate a transgenic mammalian multicellular organism. The DNA construct or the combination of DNA constructs can be introduced into the mammalian multicellular organisms by any method without limitation. For example, conventionally known techniques of gene transfer such as microinjection can be appropriately utilized.
<Step (b)>
In the step (b), alternative splicing of the specific gene in the mammalian multicellular organism is detected by detecting the expression of the reporter genes. Expression of the reporter genes can be detected by any method without limitation, and known detection methods can be appropriately utilized depending on the type of reporter gene. For example, when fluorescent proteins are used as the reporter, the expression can be detected using a fluorescence microscope or fluorescence-assisted sorter.
The DNA construct or the combination of DNA constructs contains at least two different reporter genes in total, and as described above, these reporter genes are inserted into the specific gene so that transcripts of the different reporter genes are fused with each of multiple mature mRNAs generated by alternative splicing. Therefore, by detecting the expression of these at least two different reporter genes in step (b), which form of mature mRNA is produced by alternative splicing can be checked; in other words, detection of alternative splicing patterns of the specific gene in vivo in mammalian multicellular organisms is made possible. The method for detecting alternative splicing of a specific gene in a mammalian multicellular organism also enables to detect tissue-specific and/or developmental stage-specific alternative splicing patterns in detail by changing the type of the promoter that is included in the DNA construct or the combination of DNA constructs, or changing developmental stage of the mammalian multicellular organism used.
((2) Method for Testing Whether or not a Compound to be Tested Affects Alternative Splicing of a Specific Gene)
The method for testing whether or not a compound to be tested affects alternative splicing of a specific gene of the present invention comprises the steps of (a) introducing into the mammalian multicellular organism a DNA construct in the first system or a combination of DNA constructs in the second system (each contain the specific gene and the reporter gene), (b) allowing the mammalian multicellular organism to contact the compound to be tested, (c) detecting the alternative splicing of the specific gene in the mammalian multicellular organism by detecting the expression of the reporter genes; and (d) determining whether or not the expression of the reporter genes detected in the (c) has changed compared to the expression of the reporter genes in a control which is not allowed to contact the compound to be tested.
<Step (a) and Step (c)>
The step (a) and the step (c) can be performed in the same manner as the step (a) and the step (b) in the “method for detecting alternative splicing of a specific gene in a mammalian multicellular organism” mentioned above, respectively.
<Step (b)>
In the step (b), the mammalian multicellular organism, into which the DNA construct or the combination of DNA constructs is introduced in the step (a), is allowed to contact a compound to be tested. The “compound to be tested” is not particularly limited and can be appropriately selected depending on the application from substances which one wants to test for whether or not they affect alternative splicing of the specific gene. Examples thereof include purified proteins, partially purified proteins, peptides, nonpeptidic compounds, artificially synthesized compounds, naturally-occurring compounds, and the like. In addition, the method for allowing the mammalian multicellular organism to contact the “compound to be tested” is not particularly limited; examples thereof include a method in which the “compound to be tested” is injected by microinjection, a method in which the “compound to be tested” is fed to the mammalian multicellular organism by mixing it in a food, and the like.
<Step (d)>
In the step (d), whether or not the expression of the reporter genes detected in the (c) has changed compared to the expression of the reporter genes in a control which is not allowed to contact the compound to be tested is determined. The change of the expression of the reporter genes is not particularly limited; examples thereof include replacement of the expression of one reporter gene with that of the other reporter gene, decrease or increase of the expression level of one reporter gene, and the like. If in step (d) it is determined that the expression of the reporter genes detected in step (c) has changed compared to the expression of the reporter genes in a control which is not allowed to contact the compound to be tested, it can be evaluated that the compound to be tested affects alternative splicing of the specific gene.
((3) Method for Identifying a Gene Region Affecting Alternative Splicing of a Specific Gene)
The method for identifying a gene region affecting alternative splicing of a specific gene of the present invention comprises the steps of (a) introducing into a mammalian multicellular organism a DNA construct in the first system, or a combination of DNA constructs in the second system (each contain the specific gene and the reporter gene), (b) treating the mammalian multicellular organism with a mutagen, (c) detecting the alternative splicing of the specific gene in the mammalian multicellular organism by detecting the expression of the reporter genes, (d) selecting an individual in which the expression of the reporter genes detected in step (c) has changed compared to the expression of the reporter genes in a control which is not subjected to the mutagen treatment, and (e) identifying a mutated gene region in the individual. The method allows the identification of trans-acting factors that affect alternative splicing of the specific gene.
<Step (a) and Step (c)>
The step (a) and the step (c) can be performed in the same manner as the step (a) and the step (b) in the “method for detecting alternative splicing of a specific gene in a mammalian multicellular organism” mentioned above, respectively.
<Step (b)>
In the step (b), the mammalian multicellular organism, into which the DNA construct or the combination of DNA constructs has been introduced in the step (a), is treated with a mutagen. The mutagen treatment of mammalian multicellular organisms is not particularly limited and can be performed by known methods. Examples thereof include treatment with ethyl methanesulfonate (EMS), treatment with ultraviolet light, and the like.
<Step (d)>
In the step (d), an individual, in which the expression of the reporter genes detected in step (c) has changed compared to the expression of the reporter genes in a control which is not subjected to the mutagen treatment, is selected. The change of the expression of the reporter genes is not particularly limited; examples thereof include replacement of the expression of one reporter gene with that of the other reporter gene, decrease or increase of the expression level of reporter gene, and the like.
<Step (e)>
In the step (e), a mutated gene region in the individual, in which the expression of the reporter genes has changed, is identified. The method for identifying the gene region is not particularly limited and conventionally known chromosome mapping and the like can be appropriately utilized.
((4) Method for Identifying a Region in a Specific Gene Affecting Alternative Splicing of the Specific Gene)
The method for identifying a region in a specific gene affecting alternative splicing of the specific gene of the present invention comprises steps of (a) introducing into the mammalian multicellular organism a DNA construct in the first system, or a combination of DNA constructs in the second system (each contain the specific gene and the reporter gene), wherein in the DNA construct or the combination of DNA constructs, a mutation has been introduced into the specific gene, (b) detecting the alternative splicing of the specific gene in the mammalian multicellular organism by detecting the expression of the reporter genes, and (c) determining whether or not the expression of the reporter genes detected in the (b) has changed compared to the expression of the reporter genes in a control in which a mutation has not been introduced into the specific gene. The method allows the identification of cis-acting DNA sequences that affect alternative splicing of the specific gene.
<Step (a)>
In the step (a), a DNA construct in the first system, or a combination of DNA constructs in the second system (each contain the specific gene and the reporter gene) is introduced into the mammalian multicellular organism, wherein in the DNA construct or the combination of DNA constructs, a mutation has been introduced into the specific gene. The method for introducing a mutation into the specific gene is not particularly limited and deletion, insertion, substitution, and the like can be appropriately induced in the specific gene by utilizing conventionally known techniques. In addition, the region in the specific gene into which a mutation is introduced is not particularly limited, and the region, for which one wants to evaluate whether the region affects alternative splicing of the specific gene, can be appropriately selected.
<Step (b)>
The step (b) can be performed in the same manner as the step (b) in the “method for detecting alternative splicing of a specific gene in a mammalian multicellular organism” mentioned above.
<Step (c)>
In the step (c), whether or not the expression of the reporter genes detected in the (b) has changed compared to the expression of the reporter genes in a control in which a mutation has not been introduced into the specific gene is determined. The change of the expression of the reporter genes is not particularly limited; examples thereof include replacement of the expression of one reporter gene with that of the other reporter gene, decrease or increase of the expression level of reporter gene, and the like. If it is determined that the expression of the reporter genes detected in the (b) has changed compared to the expression of the reporter genes in a control in which a mutation has not been introduced into the specific gene, the region in the specific gene into which a mutation is introduced can be identified as a region that affects alternative splicing of the specific gene.
The methods of the present invention: “(1) a method for detecting alternative splicing of a specific gene in a mammalian multicellular organism”, “(2) a method for testing whether or not a compound to be tested affects alternative splicing of a specific gene”, “(3) a method for identifying a gene region affecting alternative splicing of a specific gene”, and “(4) a method for identifying a region in a specific gene affecting alternative splicing of the specific gene, which utilize the first and second alternative splicing reporter systems developed by the present inventors, can be a very useful way for the comprehensive study of regulation mechanisms of alternative splicing in mammalian multicellular organisms.
The present invention relates to a method for constructing fluorescent alternative splicing reporter mini-genes.
In a preferable embodiment, the instant application presents a method for preparing a DNA vector for visualizing an alternative splicing pattern, the method comprising:

    
    
        (1) preparing a DNA construct of an amplified DNA region having exons a and b contained in a gene of interest;
        (2) adding attB to both ends of the DNA construct;
        (3) obtaining a recombinant of the DNA construct of (2) above and a donor vector having a gene for selection and tandemly arranged attP;
        (4) introducing a frame-shift mutation into exon a on the DNA construct of (3) above;
        (5) obtaining recombinants of (i) to (iii) below:
        (i) the DNA construct of (4) above
        (ii) a DNA vector comprising a gene for selection and a DNA having attL arranged in a direction opposing to each other at both ends of a DNA which has a structure of two kinds of reporter genes being ligated
        (iii) a DNA vector comprising a 3′ cassette DNA, a promoter, and a DNA having attR arranged in a direction opposing to each other at both ends of a DNA having a gene for selection.
    
    


In the embodiment, the gene of interest is not particularly limited and can be FGFR2 gene. For example, the exons a and b are exon 8 and 9 of FGFR2 gene. The reporter gene is not limited and can be a gene encoding a fluorescent protein.
In a preferable embodiment, the fluorescent alternative splicing reporter mini-genes can be constructed by the method set forth in FIG. 1 or 3.
2. Theoretical Background
2.1. Visualization of Alternative Splicing Patterns with Multiple Fluorescent Proteins
To clarify the regulatory mechanisms of alternative splicing in living cells, reporter mini-gene constructs containing multiple exons and introns have often been used. Splicing patterns of the mini-gene-derived mRNAs have usually been quantitatively analyzed with the ratio of reverse transcription (RT)-polymerase chain reaction (PCR) products after extracting total RNAs from transfected cells. The laborious multiple steps in analyzing the splicing patterns often caused deviation in the results and prevented high-throughput analysis of alternative splicing.
The Utilization of fluorescent alternative splicing reporters expressing fluorescent proteins has changed the situation. At initial stages, mono-chromatic, or single-color, fluorescent reporters were used as an indicator of splicing events in cultured cells. The mono-chromatic reporter mini-genes were designed to monitor proper splicing or skipping of alternative exons, and were utilized for isolation of mutant cell lines defective in the regulation of alternative splicing (Sheives, P. and Lynch, K. W. (2002). RNA 8, 1473-1481), functional screening for splicing regulatory elements (Wang, Z. et al., (2004). Cell 119, 831-845), and screening for small chemical compounds that altered splicing patterns (Levinson, N. et al., (2006). RNA 12, 925-930). The advantage of the mono-chromatic reporters is the simplicity of their structure. However, the readout of the mono-chromatic reporters may be affected by influence on gene expression such as transcription and translation.
Multi-chromatic alternative splicing reporters have overcome most of the caveats of the mono-chromatic reporters. The multi-chromatic reporter mini-genes were designed so that expression of each fluorescent protein represents a certain splicing event. The advantage of multi-chromatic fluorescent reporters is that the ratio of the expressed fluorescent proteins reflects the ratio of the mRNA isoforms, or one of the fluorescent proteins acts as an internal control of the expression level, in individual cells. The multi-chromatic reporters are therefore suitable for visualization of alternative splicing patterns in multi-cellular organisms.
The multi-chromatic reporter mini-genes can further be classified into two types, the multi- and the single-construct types. The former consists of multiple mini-genes each of which encodes a single fluorescent protein, while the latter contains two fluorescent protein cDNAs in a single mini-gene construct. Multi-construct reporters have been utilized to search for trans factors and cis-elements by flow cytometry of cultured cells (Newman, E. A. et al., (2006). RNA 12, 1129-1141), and to visualize developmentally regulated alternative splicing and further genetic analysis in C. elegans (Ohno, G. et al., (2008). Genes Dev 22, 360-374). A remarkable feature of the single-construct bi-chromatic reporters is that the two alternative mRNA isoforms, each of which encodes a single fluorescent protein, are generated from a common pre-mRNA in a mutually exclusive manner. The single-construct reporters are therefore sensitive to subtle changes in the alternative splicing patterns. The reporters have been utilized for analyzing regulatory factors (Orengo, J. P. et al., (2006). Nucleic Acids Res 34, e148), high-throughput screening for chemical compounds modifying the splicing regulation (Stoilov, P. et al., (2008). Proc Natl Acad Sci USA 105, 11218-11223), and for visualization of cell-type-specific alternative splicing in C. elegans (Kuroyanagi, H. et al., (2006). Nat Methods 3, 909-915) and mouse.
2.2. Designing Fluorescent Reporter Mini-Genes to Monitor Splicing Patterns
Here the present inventors show typical structures of multi-chromatic alternative splicing reporter mini-genes (FIG. 2) which the present inventors constructed as described in Examples and explain how expression of each fluorescent protein reports a specific alternative splicing event. Please notice that the reporter mini-genes described in this specification are just a few examples of possible alternative splicing reporters. Each reporter can be flexibly designed depending on alternative splicing events of interest to be visualized. An ideal mini-gene should be designed so that expression of a specific fluorescent protein unambiguously indicates a specific mRNA isoform or a specific alternative splicing event (Kuroyanagi, H. et al., (in press). Visualization and genetic analysis of alternative splicing regulation in vivo using fluorescence reporters in transgenic Caenorhabditis elegans. Nat. Protoc.).
As described above, the mini-genes in FIG. 2 can be divided into the multi-construct type (FIG. 2A, B) and the single-construct type (FIG. 2C, D). The choice of the reporter type depends on organisms and the method of mini-gene transfer. Transgenic worms generated by a standard microinjection method carry hundreds of copies of plasmid DNAs as an extra-chromosomal array (Mello, C. C. et al., (1991). EMBO J. 10, 3959-3970) and, therefore, it is generally assumed that injecting a mixture of several different mini-genes with the same vector backbone results in proportional incorporation of all the constructs in the extra-chromosomal array. One of the advantages of the multi-construct reporters is that the number of co-transferred mini-genes can be increased to more than two as described in section 4. For situations where the copy number of transferred mini-genes is small or variable, single-construct reporters might be preferable.
FIG. 2A shows schematic structure of a pair of reporter mini-genes for mutually exclusive exons. A genomic fragment of interest, from the upstream constitutive exon through the downstream constitutive exon, is placed downstream of a common promoter and a constitutive intron, followed by a cDNA for either of two fluorescent proteins and a 3′ cassette. An in-frame translation initiation codon is artificially introduced at the 5′ end of the genomic fragment. A termination codon is artificially introduced in one of the two alternative exons in each construct. From the mini-genes shown in FIG. 2A, a GFP-fusion protein is produced from an mRNA isoform in which exon a alone is included and an RFP-fusion protein is produced from an mRNA isoform in which exon b alone is selected.
FIG. 2B shows schematic structures of a pair of reporter mini-genes to monitor inclusion and skipping of a cassette exon. The order and composition of the fragment cassettes are as those in FIG. 2A. In the case shown in FIG. 2B, the length of the cassette exon is not a multiple of three bases and therefore inclusion of the cassette exon changes the reading frame of the downstream exon. The GFP cDNA is connected in frame when the cassette exon is included and the RFP cDNA is connected in frame when the cassette exon is excluded.
FIGS. 2C and 2D show schematic structures of single-construct bi-chromatic reporters. These constructs rely on an unusual feature of some fluorescent protein cDNAs in which an alternate reading frame lacks a termination codon (Orengo, J. P. et al., (2006). Nucleic Acids Res 34, e148). In the cases shown in FIGS. 2C and 2D, the RFP and GFP cDNAs are connected in a different reading frame so that translation of the alternate frame of RFP cDNA leads to generation of a fluorescent protein from GFP cDNA. When a fluorescent protein is generated from RFP cDNA, translation will be ceased at its own termination codon. The mini-gene shown in FIG. 2C is for monitoring inclusion and skipping of a cassette exon. GFP cDNA is in frame when the cassette exon is included and RFP cDNA is in frame when the cassette exon is excluded. The mini-gene shown in FIG. 2D is for monitoring the selection of mutually exclusive exons. In this case, one nucleotide is inserted into exon a to cause a frame-shift when this exon is selected. Glutathione S-transferase (GST) of E. coli is used as an N-terminal tag for expression of the fusion proteins. The GFP-fusion protein is produced when exon a alone is included and RFP-fusion protein is produced when exon b alone is selected. Neither of the fluorescent proteins is produced when both exons are included or skipped.
2.3. Constructing Fluorescent Reporter Mini-Genes
The present inventors construct fluorescent alternative splicing reporter mini-genes by site-specific recombination utilizing MULTISITE GATEWAY® vector construction system (Invitrogen). Major advantage of homologous recombination in mini-gene construction is that ‘Expression’ vectors with a variety of structures, as described above, can be easily and rapidly constructed by assembling modular DNA fragments cloned in ‘Entry’ and ‘Destination’ vectors. In this section, the present inventors focus on practical use of the MULTISITE GATEWAY® vector construction system and other aspects to be considered in designing fluorescent reporter mini-genes.
23.1. MULTISITE GATEWAY® Vector Construction System
The MULTISITE GATEWAY® vector construction system uses site-specific recombinational cloning to allow simultaneous cloning of two, three or four separate DNA fragments of interest in a defined order and orientation. FIG. 3 schematically illustrates construction of an ‘Expression’ clone by performing a ‘2-fragment’ recombination reaction. Genomic DNA fragments of interest are cloned in ‘Entry’ vectors (FIG. 3A) and the fragments are assembled between homologous recombination sites of the ‘Destination’ vectors (FIG. 3B). A key feature of the MULTISITE GATEWAY® vector construction system is that five sets of modified att sites have an orientation and demonstrate the specificity of homologous recombination as in the standard Gateway system: for example, attBI site reacts only with attP I site, but not other attP sites, to generate attL1 and attRI sites in ‘BP’ reaction (FIG. 3A), and attL5 site reacts only with attR5 site, but not other attR sites, to generate attB5 and attP5 sites in ‘LR’ reaction (FIG. 3B). For more details about the MULTISITE GATEWAY® vector construction system and ‘3-fragment’ and ‘4-fragment’ recombination reactions, please refer to the provider's website.
All the att sites in our reporter mini-genes, or ‘Expression’ clones, are attB sites and reside within exons (FIGS. 2 and 3). The attB sequences (21 to 25 base pairs) are the shortest stretches among all att sites. The present inventors have not experienced cryptic splicing within the attB sequences in C. elegans or mammalian cells. It is recommended to use a fixed reading frame in the attB sequences and the present inventors usually do so (see section 3). As attB1, attB5 and attB2 sequences lack ATG and a termination codon in any frames, they can theoretically be used in any frames.
2.3.2. Other Aspects to be Considered in Mini-Gene Construction
mRNAs with premature termination (nonsense) codons (PTCs) are selectively degraded by a quality-control mechanism called nonsense-mediated mRNA decay (NMD). In mammals, NMD is considered to be induced when an exon junction complex (EJC), a protein complex deposited upstream of exon-exon boundaries after RNA splicing, resides downstream of the termination codon in the first round of translation (Chang, Y. F. et al., (2007). Annu Rev Biochem 76, 51-74, Isken, O. and Maquat, L. E. (2007). Genes Dev 21, 1833-1856). It is critical to design the fluorescent reporter mini-genes so that the mRNA isoforms encoding the fluorescent proteins escape NMD. As the GFP and RFP cDNAs reside in the last exon in mini-genes shown in FIG. 2, the productive isoforms from these mini-genes would escape NMD in mammals. In C. elegans (Kuroyanagi, H. et al., (2007). Mol Cell Biol 27, 8612-8621, Pulak, R. and Anderson, P. (1993). Genes Dev 7, 1885-1897) and yeast (Kebaara, B. W. and Atkin, A. L. (2009). Nucleic Acids Res 37, 2771-2778, Amrani, N. et al., (2004). Nature 432, 112-118), long 3′ untranslated region (UTR) triggers NMD independent of exon-exon boundaries, and therefore the mRNA isoforms encoding RFP proteins in FIGS. 2C and 2D may be degraded by NMD in these organisms.
Genomic fragments utilized in the mini-gene constructs usually undergo proper splicing. However, trimming of constitutive exons and/or deletion of long intronic regions may lead to inefficient splicing or deregulation of alternative splicing. Repeated minigene optimization may be required to establish a reporter reflecting the alternative splicing pattern of the endogenous gene. The present inventors have not experienced cryptic splicing in GFP or RFP cDNAs, but other cDNAs for N-terminal and C-terminal tags may serve as cryptic splice sites.
Amino acid sequences derived from the gene of interest may greatly affect folding, stability and/or subcellular localization of the fluorescent fusion proteins. It is therefore critical to predict the property of the fusion proteins in designing the mini-genes. Various N-terminal tags such as GST (FIG. 2D) may stabilize the expression of the fusion proteins and improve the result. It is also critical to force translation initiation at the designed initiation codon. ATG codons in the exonic regions and in the N-terminal tags may be the cause of aberrant translation initiation and reduce the production of the fluorescent proteins.
Several groups have generated reporter system to visualize splicing regulation of FGFR2 gene. Newman et al. generated fluorescent reporter vectors with the minimum genomic region that can reflect endogenous splicing regulation in AT-3 and DT-3 cells. With this reporter, they identified specific ISEs, including ISS/ISS-3, that respond to Fox regulation [Newman E A, Muh S J, et al. (2006) Rna 12: 1129-1141]. Bonano et al. constructed another fluorescent reporter vector by using the genomic fragment around exon 8, and they visualized the regulation of exon 8 inclusion in the mouse [Bonano V I, et al. (2006) Rna 12: 2073-2079]. In this invention, the present inventors originally developed a transgenic reporter system using FGFR2 gene as model and succeeded in visualizing tissue-specific expression profiles of the mutually exclusive exons through differential expression of EGFP and RFP in mice for the first time. Using the mostly entire genomic region around alternative exons with their upper and lower constitutive exons, the present inventors could evaluate which splice site sequences and cis-elements are truly critical for regulations. This system has great advantage, 1) to monitor the dynamism of splicing regulation in vivo with single cell resolution, 2) to identify essential cis-elements and trans-factors, and reveal the hidden mechanisms of splicing regulation like this study, 3) to identify the essential candidate factors by fluorescent color change or to screen the regulators using cDNA or siRNA library. In this way, splicing reporter system has great advantage to decipher the hidden splice code and also to reveal the important roles regulated by alternative splicing during development or in adult with tissue- or cell-type specific manner.
With splicing reporter system, the present inventors showed that two mutually exclusive exons of FGFR2 gene seem to have “primary” and “secondary” fates. Without tissue-specific regulators, “primary” exon 9 is chosen as the “default” and the “secondary” exon 8 is ignored depending on the relative strength of their 3′ splice sites (FIG. 12A, Non-Epithelial or Mesenchymal regulation). When tissue-specific key regulator ESRPs are expressed, these factors bind close to the 3′ splice site of “primary” exon 9 with Fox, and repress exon 9 recognition (FIG. 12A, Epithelial regulation). Mutually exclusive alternative splicing regulated through the ordered splicing recognition with tissue specific factors has been observed in nematodes [Ohno G, et al. (2008) Genes Dev 22: 360-374]. With this ordered splice-site recognition, transacting regulators can sequentially control the selection of mutually exclusive exons in a tissue-specific manner, in a similar manner to which a chess piece, usually a knight, can simultaneously attack a rook and check the king so that the former must be lost (FIG. 12A). This regulatory mechanism clearly answered to our initial questions described above. So far, tissue-specific alternative splicing regulation has been understood from the viewpoint of the balance between enhancement and silencing of the alternative exons mediated by trans-factors through specific cis-elements [Matlin A J, et al. (2005) Nat Rev Mol Cell Biol 6: 386-398.]. In this idea, each alternative exon is regulated discretely, and the final splicing isoforms are determined from the sum and balance of these independent regulations. If this were the case in mutually exclusive alternative splicing, the initial splicing products would be mixtures of the single-exon-inclusion form, double-inclusion form, and double-skip form. As the last two isoforms would be eliminated by NMD [Chang Y F, et al. (2007) Annu Rev Biochem 76: 51-74], only the single-exon-inclusion forms would remain as final splicing products. On the other hand, if mutually exclusive splicing is regulated by the switch-like manner, the initial spliced products would consist mainly of one of the single-inclusion forms. If the exon selection in AT-3 cell is on a balance of combined regulations of enhancer for exon 9 and repressor for exon 8, overexpression of repressor for exon 9 should produce mostly the double-skip form. The present inventors did not, however, observe this (FIG. 10A). In addition, when the 3′ slice site of exon 8 in the reporter was changed to be as strong as that of exon 9, the double-inclusion splicing product became dominant in AT-3 cell, without any change in the transacting factors or specific cis-elements affecting exon 8 (FIG. 7, lane 2). Also, splice site mutation of exon 9 caused switching to epithelial exon 8 in non-epithelial AT-3 cell (FIG. 8, lane 2 and 4). These results suggest that alternative splicing is not achieved by a complicated balance of multiple regulators, but is defined by a simple switch-like mechanism in which only one appropriate exon can be selected and switched by key regulators.
The present inventors' previous study showed that mutually exclusive splicing of the worm FGFR homologue gene egl-15 is regulated by the cooperation of broadly expressed Fox-1/ASD-1 family and the muscle-specific RNA binding motif proteins (RBMs) of SUP-12 (a nematode homologue of mRBM24), which act together to repress inclusion of alternative exon 5B to promote muscle-specific expression of exon 5A [Kuroyanagi H, et al. (2006) Nat Methods 3: 909-915, Kuroyanagi H, et al. (2007) Mol Cell Biol 27: 8612-8621.](FIG. 12B, nematode). In the case of mammalian FGFR2, the generally expressed Fox family cooperate with the tissue-specific factor ESRPs (RBM35a & b) to repress alternative inclusion of exon 9 and to promote epithelial tissue-specific expression of exon 8 (FIG. 12B, mammals). Moreover, both of nematode and mammalian Fox family proteins bind to the UGCAUG element, and SUP-12 and ESRPs bind to GU stretches. Nematode has rather similar number of genes ( ̃20,000) comparing with mammals but the size of its genome is much smaller (about 1/30) with very shorter intron (average 561 bp). So, alternative splicing in nematode is considered to be regulated with much simpler rules [Zahler A M (2005) WormBook: 1-13, Kabat J L, et al. (2006) PLoS Comput Biol 2: e86.]. Thus, it is amazing that regulatory mechanism of tissue-specific alternative splicing is evolutionally conserved from nematode to mammals, and regulation though Fox and another tissue-specific RBMs may turn out to be a widespread essential phenomenon to regulate tissue-specific alternative splicing of multiple genes. The present inventors' findings also provide the significant cue to understand how a number of spliced genes are regulated in various tissue-specific manners by a limited number of regulators, eventually to understand seemingly complicated developmental process or tissue-specific functions.
All prior art documents cited in the specification are incorporated herein by reference.
EXAMPLES
Examples of the present invention are illustrated below, but these are not to be construed as limiting the present invention.
Example 1
Protocol
1. Constructing Genomic DNA Fragment Cassettes in ‘Entry’ Vectors
The present inventors perform the ‘BP’ reaction to clone genomic fragments of interest in ‘Entry’ vectors of the MultiSite Gateway system. To amplify attB-flanked genomic fragments, the present inventors usually perform a two-step PCR procedure. The first PCR is performed with primers that are gene-specific and contain a part of the attB sequences at their 5′ ends. The first PCR product is then used as a template for the second PCR with attB adapter primers. The advantages of the two-step PCR procedure are that the gene-specific primers (GSPs) to be synthesized would be shorter and that the attB adapter primers can be used repeatedly for cloning other DNA fragments in different mini-gene projects. Here the present inventors demonstrate how to construct ‘Entry’ clones for ‘2-fragment’ recombination reaction as schematically shown in FIG. 3A. The genomic DNA fragment is cloned in either of the ‘Entry’ vectors depending on the design of the mini-genes to be constructed (see section 2.2). The ‘3-fragment’ and ‘4-fragment’ recombination reactions may also work in mini-gene construction, although they are less efficient and we have few experiences.
1.1. Primer Design
The GSPs must have 12 bases of the attB site on the 5′ end followed by 18 to 25 bases of template- or gene-specific sequences (Table 1; Sequences of primers used for constructing ‘Entry’ clones). Kozak's consensus sequence can be inserted between the attB1 and the gene-specific sequences to force translation initiation as shown in Table 1. Termination codons must be included in or excluded from the reverse GSPs, according to the design of the reporter mini-genes. If the DNA fragment is designed to be fused with N- and/or C-terminal tags, the GSPs must be carefully designed to maintain the proper reading frame in the attB sequences as indicated in Table 1.
The attB adapter primers for the second PCR consist of the following common structure: four guanine (G) residues at the 5′ end followed by a 22- or 25-base complete attB sequence (Table 1).








TABLE 1







GSP-attB1F
5′-AA AAA GCA GGC TNN -(gene-specific sequence)-3′ (SEQ ID



NO: 1)



* To avoid generating a stop codon, NN cannot be AA, AG, or GA.


 


GSP-attB1F
5′-AA AAA GCA GGC TCC ACC ATG G -(gene-specific


(with ATG)
sequence)-3′ (SEQ ID NO: 2)



* Kozak consensus sequence allows efficient protein expression in



eukaryote cells.


 


GSP-attB5R
5′-T ATA CAA AGT TGT -(gene-specific sequence)-3′ (SEQ ID



NO: 3)


 


attB1adapterF
5′-GGGG ACAAGTTTGTACAAAAAAGCAGGCT-3′ (SEQ ID



NO: 4)


 


attB5adapterR
5′-GGGG ACAACTTTTGTATACAAAGTTG-3′ (SEQ ID NO: 5)


 


GSP-attB5F
5′-AT ACA AAA GTT G -(gene-specific sequence)-3′ (SEQ ID



NO: 6)


 


GSP-attB2R
5′-A GAA AGC TGG GT -(gene-specific sequence)-3′ (SEQ ID



NO: 7)


 


attB5adapterF
5′-GGGG ACAACTTTGTATACAAAAGTTG-3′ (SEQ ID NO: 8)


 


attB2adapterR
5′-GGGG ACCACTTTGTACAAGAAAGCTGGGT-3′ (SEQ ID



NO: 9)









Underlines indicate 12 bases of the attB sequences included in the GSPs.
1.2. Performing PCR
The PCRs should be performed with a proofreading polymerase, such as PRIMESTAR® HS DNA Polymerase (TaKaRa). The annealing temperature of the second PCR should be 45 degrees C. because the annealing sequences are just 12 base pairs.
Protocol 1: Two-Step PCR Amplification of attB-DNA Fragments
1. Perform the first PCR in a 25 microliter (microL) mixture containing standard reagents with 0.2 microM each of GSPs. The conditions of the PCR should be optimized depending on the amount of the template and the size of the fragment to be amplified. Check the PCR product by standard agarose gel electrophoresis.
2. Prepare 50 microliter (microL) of the second PCR mixture containing standard reagents and 0.3 microM each of attB adapter primers. Add the mixture to 10 microliter (microL) of the first PCR reaction mixture and perform 5 cycles of PCR with annealing at 45 degrees C. Check by agarose gel electrophoresis that the amount of the PCR product has increased in the second PCR.
3. Optionally, add 1 microliter (microL) Dpn I* (New England Biolabs) and incubate at 37 degrees C. for 1 hour to destroy template DNA. *If the PCR template is a plasmid containing the kanamycin-resistance gene, the PCR mixture should be treated with Dpn I before purifying the attB-PCR products. Dpn I recognizes methylated GATC sites in bacteria-derived DNA. Dpn I treatment greatly reduces background in the ‘BP’ recombination reaction associated with template contamination.
4. Purify the attB-PCR product with a standard DNA purification column.
1.3. ‘BP’ Recombination Reaction and Selection of ‘Entry’ Clones
Perform ‘BP’ recombination reaction between each attB-flanked DNA fragment and an appropriate attP-containing ‘Donor’ vector (Table. 2: Selection of ‘Donor’ vectors for constructing ‘Entry’ clones by ‘BP’ reaction) to generate an ‘Entry’ clone.









TABLE 2







pENTR Vectors


pDONR Vectors
DNA fragments to be cloned
generated







pDONR 221 P1-P5r
attB1F/attB5R-flanked
pENTR-L1-R5



PCR products


pDONR 221 P5-P2
attB5F/attB2R-flanked
pENTR-L5-L2



PCR products









Protocol 2: BP CLONASE II® Enzyme Mix Reaction and Selection of Appropriate ‘Entry’ Clones
1. Add the following components to a 1.5-ml microcentrifuge tube and mix: attB-PCR product (15 to 150 ng), pDONR vector (supercoiled, 75 ng), and deionized distilled water (DDW) or TE to 4 microliter (micro L). Add 1 microliter (microL) BP CLONASE II® enzyme mix (Invitrogen) to the components above and mix well by briefly vortexing or tapping.
2. Incubate the ‘BP’ reaction mixture at room temperature or at 25 degrees C. for 1 hour or overnight. We usually omit proteinase K digestion.
3. Transform E. coli strain DHSalpha or others* with 1 to 3 microliter (microL) of the reaction mixture and select for kanamycin-resistant ‘Entry’ clones. Check the sequence of the insert. *E. coli strains with F′ episome (e.g. TOP10F′) cannot be used for transformation to select ‘Entry’ clones. These strains contain the ccdA gene and will prevent negative selection with the ccdB gene in the pDONR vectors.
1.4. Modification of ‘Entry’ clones (Optional)
According to the design of the mini-genes, introduce a termination codon or a frame-shift into exons, and/or modify or disrupt putative cis-elements of the ‘Entry’ clones. Sequences of the genomic fragment should be carefully modified to avoid disruption of putative cis-regulatory elements. The present inventors introduce termination codons or frame-shifts in less conserved stretches among related species. The present inventors utilize Quickchange II or Quickchange II XL (Stratagene) for the site-directed mutagenesis. The present inventors usually sequence the entire insert after mutagenesis.
2. ‘LR’ Recombination Reaction and Selection of ‘Expression’ Clones
Gateway ‘Destination’ vectors usually provide a promoter and a 3′ cassette (FIG. 2). A variety of ‘Destination’ vectors for expression in cultured cells are commercially available. ‘Destination’ vectors can also be constructed by ligation-based insertion of Destination vector cassettes (Invitrogen) into existing vectors containing the ampicillin-resistance gene. The present inventors performed ‘BP’ reaction to convert existing expression vectors into ‘Destination’ vectors, which were used in our previous studies [Ohno, G., et al., (2008) Genes Dev 22, 360-374, Kuroyanagi, H. et al., (2006) Nat Methods 3, 909-915, Kuroyanagi, H. et al., (2007) Mol Cell Biol 27, 8612-8621.]. The present inventors can convert any existing vectors containing the ampicillin-resistance gene into ‘Destination’ vectors at desired positions with desired frames. Details of the conversion method will be described elsewhere [Kuroyanagi, H. et al., (in press) Nat. Protoc.]. Nucleotide sequences of the ‘Destination’ vectors the present inventors constructed for expression in C. elegans are available on the C. elegans Promoter/Marker Database (http://www.shigen.nig.ac.jp/c.elegans/promoter/index.jsp).
The present inventors have constructed a variety of ‘Entry’ clones of fluorescent protein cassettes in pENTR-L1-R5 and pENTR-L5-L2 vectors with or without initiation and/or termination codons.
Protocol3: LR CLONASE II PLUS® Enzyme Mix Reaction and Selection of Appropriate ‘Expression’ Clones.
1. Add the following components to a 1.5-ml microcentrifuge tube and mix: Destination′ vectors (75 ng), ‘Entry’ clones (15 to 100 ng each), DDW or TE to 4 microliter (microL). Add 1 microliter (microL) LR CLONASE II PLUS® enzyme mix (Invitrogen) to the components above and mix well by briefly vortexing or tapping.
2. Incubate the ‘LR’ reaction mixture at 25 degrees C. or at room temperature overnight. The present inventors usually omit the proteinase K digestion.
3. Transform E. coli strain DHSalpha or others* with 1 to 3 microliter (microL) of the reaction mixture and select for ampicillin/carbenicillin-resistant ‘Expression’ clones. The present inventors routinely check resistance of the ampicillin/carbenicillin-resistant colonies to ampicillin/carbenicillin, chloramphenicol and kanamycin. *E. coli strains with F′ episome cannot be used to select ‘Expression’ clones.
4. Select clones that are resistant only to ampicillin/carbenicillin, check restriction enzyme digestion patterns of the mini-prep plasmid DNAs and sequence the boundaries of the DNA fragments.
3. Transfection of Cultured Cells and Generation of Transgenic Animals.
Transient transfection of cultured cells with the fluorescent reporter mini-gene(s) is performed using standard transfection reagents. Expression of the fluorescent proteins can be analyzed by utilizing standard compound microscopes, flow cytometry and other instruments.
Transgenic animals can be generated by standard methods. Expression of the fluorescent proteins in mice can be analyzed by observing sections under compound microscopes or confocal microscopes. The present inventors usually utilized high-magnification fluorescence dissection microscopes or confocal microscopes to analyze cell- and tissue-specific expression of the fluorescent reporter proteins in transgenic worms.
4. Checking Splicing Pattern of the Mini-Gene-Derived mRNAs.
The present inventors strongly recommend analyzing splicing patterns of the mini-gene-derived mRNAs to confirm that the reporter mini-genes are expressed and properly spliced and that the alternative splicing pattern is as expected from that of the endogenous gene and is consistent with the ratio of the expressed fluorescent proteins.
Protocol 4: RT-PCR Analysis of Mini-Gene-Derived mRNAs
1. Total RNA is extracted from cells, tissues or organisms by utilizing RNEASY MINI® extraction kit (QIAGEN) or equivalents and DNase I following manufacturer's instructions.
2. In RT, 1 to 2 microgram of the total RNA is reverse transcribed with PrimeScript (TaKaRa) or Superscript II (Invitrogen) and oligo(dT) as a primer following manufacturers' instructions.
3. For PCR, the present inventors usually use nonproofreading polymerases such as Ex Taq (TaKaRa) and BIOTAQ® polymerase (BIOLINE), and a mini-gene-specific primer set. As GFP-specific reverse primers, the present inventors use 5′-TGTGGCCGTTTACGTCG-3′ (SEQ ID NO: 10) or 5′-TTTACTTGTACAGCTCGT-3′ (SEQ ID NO: 11). As mRFPspecific reverse primers, the present inventors use 5′-GGAGCCGTACTGGAACTGAG-3′ (SEQ ID NO: 12) or 5′-TTAGGCGCCGGTGGAGTG-3′ (SEQ ID NO: 13). attB adapter primers can also be used.
4. To analyze RT-PCR products, directly sequence the purified products, or clone the products in TA-vectors such as PGEM-T EASY® (Promega) and sequence them.
Example 2
Examples of Experiments
FIGS. 4A and 4B show an example of visualizing tissue-specific alternative splicing of mutually exclusive exons in C. elegans. FIGS. 4C and 4D show an example of visualizing viral infection-induced intron retention in HeLa cells [Nojima, T. et al., (2009) Nucleic Acids Res 37, 6515-6527.]. FIGS. 4E and 4F show an example of visualizing mutually exclusive alternative splicing in mice.
Formal names of the abbreviations used in the present specification can be shown below.
ABBREVIATIONS


    
    
        EJC, exon junction complex;
        GFP, green fluorescent protein;
        GSP, gene-specific primer;
        GST, glutathionine S-transferase;
        mRNA, messenger RNA;
        NMD, nonsense-mediated mRNA decay;
        PCR, polymerase chain reaction;
        PTC, premature termination codon;
        RFP, red fluorescent protein;
        RT, reverse transcription;
        UTR, untranslated region.
    
    


MATERIALS AND GLOSSARY 








TABLE 3







pENTR-L1-R5
These are vector backbones of the ‘Entry’


and
vectors and the maps are schematically drawn in


pENTR-L5-L2
“1-page summary” and FIG. 2A, 2B.


nonsense-
An mRNA quality-control mechanism that degrades


mediated mRNA
abnormal mRNAs with premature termination codons


decay (NMD).
arising from genetic mutations or a consequence



of mistakes in gene expression.


exon junction
A complex of proteins deposited as a consequence


complex (EJC),
of pre-mRNA splicing ~20-24 nucleotides upstream



of splicing-generated exon-exon junctions of newly



synthesized mRNA.


TaKaRa
Seta 3-4-1, Otsu, Shiga 520-2193, Japan



http://www.takara-bio.com/


BIOLINE
London, UK www.bioline.com









Materials & Methods
Plasmid Construction
The present inventors constructed the reporter vector essentially as described in the text. The FGFR2 genomic region spanning from exon 7 to exon 10 from mouse genomic DNA was amplified by PCR and cloned into GATEWAY DESTINATION® Vector (Invitrogen), carrying both EGFP (Clontech) and mRFP [Campbell R E, et al. (2002) Proc Natl Acad Sci USA 99: 7877-7882.] with different reading frames, under the control of the CAGGS promoter. To stabilize and enhance reporter protein expression, artificial sequence of modified glutathione S-transferase (GST) gene (QIAGEN) was introduced in front of the FGFR2 genomic fragment and connected in frame with exon 7. A I-kbp fragment at the center of the FGFR2 intron 9 was removed because of its significant reducing effect in mRNA expression. Mutations of cis-elements were introduced by using QUIKCHANGE XL II® (Stratagene). Mouse Fox 1 and Fox2 cDNAs were kindly provided by Dr. Kawamoto [Nakahata S, Kawamoto S (2005) Nucleic Acids Res 33: 2078-2089.] and cloned into the GATEWAY DESTINATION® vector driven by a CAGGS promoter. Primer sequences for amplifying FGFR2, deleting 1 kbp in the middle of intron 9, and introducing mutations were indicated below.
Primer sequences for amplifying FGFR2, deleting 1 kbp in the middle of intron 9, and introducing mutations;







FGFR2 amplify-F:


(SEQ ID NO: 14)


ggctgccctacctcaaggtcctg


 


FGFR2 amplify-R:


(SEQ ID NO: 15)


ctctctcacaggcgctgggttgcag


 


intron 9 partial deletion-F:


(SEQ ID NO: 16)


gcatgccttgatagagtggcctctc-ctgttgaaccttcccctggag


 


intron 9 partial deletion-R:


(SEQ ID NO: 17)


ctccaggggaaggttcaacag-gagaggccactctatcaaggcatgc


 


exon 9 mutation for deleting PTC-F:


(SEQ ID NO: 18)


gaggttctctatattcggaatgtTacttttgaggatgctggg


 


exon 9 mutation for deleting PTC-R:


(SEQ ID NO: 19)


cccagcatcctcaaaagtAacattccgaatatagagaacctc


 


exon 9 3′ splice site mutation-F:


(SEQ ID NO: 20)


gcttcgtttgttttctctgccgccggtgttaacaccacggac


 


exon 9 3′ splice site mutation-R:


(SEQ ID NO: 21)


gtccgtggtgttaacaccggcggcagagaaaacaaacgaagc


 


exon 9 5′ splice site mutation-F:


(SEQ ID NO: 22)


ctgcatggttgacagttctgccaccaacatactgctctttctctc


 


exon 9 5′ splice site mutation-R:


(SEQ ID NO: 23)


gagagaaagagcagtatgttggtggcagaactgtcaaccatgcag


 


TGCATG mutation-F:


(SEQ ID NO: 24)


gggccaatttttccatgtgttcaatttacgtacgttctaggtggtgacg


 


TGCATG mutation-R:


(SEQ ID NO: 25)


cgtcaccacctagaacgtacgtaaattgaacacatggaaaaattggccc


 


ISE/ISS-3 mutation-F:


(SEQ ID NO: 26)


taggtggtgacgccgaatctcctgatggcc


 


ISE/ISS-3 mutation-R:


(SEQ ID NO: 27)


ggccatcaggagattcggcgtcaccaccta






Generation of FGFR2 Splicing Reporter Mice
The constructed FGFR2 splicing reporter vector was linearized and injected into the pronucleus of a C57BL/6 oocyte. Mice were genotyped by PCR with primers for EGFP using genomic DNA from the postnatal and late embryonic tails, or yolk sacs from earlier embryos. All experiments were performed in accordance with the protocols certified by the Institutional Animal Care and Use Committee of the Tokyo Medical and Dental University (Approval Numbers #0070220, #0080179, and #0090084).
Cell Culture and Transfection
Rat AT-3 and DT-3 prostate carcinoma cell lines were kindly provided by Dr. Garcia-Blanco and Dr. Carstein. Cells were maintained in Dulbecco's modified eagle medium (D-MEM) with 10% fetal calf serum (FCS), and vectors were transfected with TRANSFECTIN® reagent (BIORad). Stealth siRNA was used in knockdown experiments on endogenous Fox2 and was transfected using LIPOFECTAMINE® RNAiMAX reagent (Invitrogen).
Microscopy
Fluorescent images of whole embryos of reporter transgenic mice were capture under a fluorescence microscope (MZ16FA, Lecia) with a charge-coupled device (CCD) camera (DP71, Olympus). The present inventors also used a confocal microscope (Fluoview FV1000, Olympus) to capture fluorescent images of sectioned transgenic embryos, and the captured images were processed by means of METAMORPH® software (Molecular Devices). Fluorescent images of cultured cells and bright-field images showing in situ hybridization were captured by using a Nikon Eclipse E600 microscope with a CCD camera (DP71, Olympus).
RT-PCR
Total RNAs and RT-PCR were performed as described previously [Kuroyanagi H, et al., (2006) Nat Methods 3: 909-915, Takeuchi A, et al. (1998) Eur J Neurosci 10: 1613-1620.]. The identity of all splicing variants was confirmed by sequencing. Amounts of PCR products were measured with a 2100 BioAnalyzer with Agilent DNA1000 kits (Agilent Technology), and the quantitative analyses were performed in more than three independent experiments.
Primer sequences used in the RT-PCR assays are listed below.
Primer sequences used in the RT-PCR assays;









Reporter FGFR2-F



(SEQ ID NO: 28)



5′-GGCCTTTGCAGGGCTGGC-3′



 



Reporter FGFR2-R



(SEQ ID NO: 29)



5′-GGAGCCGTACTGGAACTGAGG-3′



 



Fox1-F



(SEQ ID NO: 30)



5′-GGCACCGCCACACAGACAGATGA-3′



 



Fox1-R



(SEQ ID NO: 31)



5′-TCCTGGTTGGCCTGGCACAACAG-3′



 



Fox2-F



(SEQ ID NO: 32)



5′-CAACAACTCCTGACGCAATGGTTCAGC-3′



 



Fox2-R



(SEQ ID NO: 33)



5′-GATTTTACGGCCCTCTACCACGGTG-3′



 



ESRP1-F



(SEQ ID NO: 34)



5′-CAGGAGATGCCTTTATCCAGATGAAGTC-3′



 



ESRP1-R



(SEQ ID NO: 35)



5′-CAGTATTGTAGGCCAGGCCCTG-3′



 



ESRP2-F



(SEQ ID NO: 36)



5′-CCTACACAGCCACCATTGAAGACATTC-3′



 



ESRP2-R



(SEQ ID NO: 37)



5′-GGTGAGGTAGCCCACAGTAGTG-3′






In Vitro Exon-Recognition Assay
The PCR products of T7-ex 9 wt (mouse FGFR2 intron 8, 200 nt; exon 9, 145 nt; and intron 9, 105 nt), T7-ex 9 containing UGCAUG and ISE/ISS-3 in intron 8 (intron 8, 237 nt; exon 9, 145 nt; and intron 9, 74 nt), and T7-ex 8 wt (intron 8, 200 nt; exon 8, 149 nt; and intron 9, 100 nt) were used as DNA templates for T7 transcription. A mutated DNA template of 3′ ss in intron 8 and 5′ ss in intron 9 was prepared from mutated reporter vectors as shown in FIG. 3A. The RNA substrates were labeled with 32P by in vitro T7 transcription. In vitro splicing reaction and UV cross linking were performed under the conditions described by Sawa [Sawa H, Shimura Y (1992) Genes Dev 6: 244-254.]. To identify the shifted bands by UV cross linking, pre-heated cDNA oligo (10 microgram/mL) for U1 (5′-CGGAGTGCAATG-3′/SEQ ID NO:38) or U2 (5′-CAGATACTACACTTG-3′/SEQ ID NO:39) was added to the RNAs after UV cross linking, and the U1 or U2 cDNA oligo/RNAs mixture were digested with 50 U/mL of RNase H at 30 degrees C. for 10 min. In FIG. 5C, highly purified Flag-Fox2 and Flag-ESRP1 were added to RNAs and incubated at room temperature for 5 min before splicing reaction to examine their inhibitory or activating effects on exon recognition of U1 snRNA or U2 snRNA. After these reactions, RNAs were subjected to denaturing PAGE analysis and autoradiography.
In Situ Hybridization
In situ hybridization was carried out as previously described [Takeuchi A, et al. (2003) Nat Genet. 33: 172-176, Takeuchi A, O'Leary D D (2006) J Neurosci 26: 4460-4464.], with modifications. Briefly, embryos were fixed with 4% paraformaldehyde, cryo-protected with 30% sucrose, embedded in optimal cutting temperature (OCT) compound, and cut into sections in 20 micrometer thickness. Antisense RNA probes labeled with digoxigenin were visualized with Fab fragments from an antibody against digoxigenein conjugated with alkaline phosphatase (Roche) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/Nitroblue Tetrazolium (NBT) solutions. Sections were counterstained with Methyl Green. The following cDNA was used as the riboprobe: Fox1 (131-865 bp from mouse cDNA), Fox2 (2261-2991 bp from mouse cDNA), ESRP1 (1168-1708 bp from mouse cDNA), and ESRP2 (208-807 bp from mouse cDNA).
Example 3
Generation of the FGFR2 Splicing Reporter System
The present inventors used a 3.7-kb genomic fragment of FGFR2 gene that included two alternative exons (exon 8 and 9) flanked by their upstream and downstream exons (exon 7 and 10), with introns in between (FIG. 6A). By using mostly entire genomic region around alternative exons, this reporter system was expected to contain all the regulatory cis-elements essential for tissue-specific regulation and to tell which splice site sequences and cis-elements are truly critical for regulations. The genomic fragment was cloned into a vector containing RFP and EGFP in tandem with different reading frames [Orengo J P, et al., (2006) Nucleic Acids Res 34: e148.]. With this reporter system, splicing regulation could be monitored from a single reporter vector that expresses either EGFP when exon 8 is chosen or RFP when exon 9 is selected (FIG. 6A).
By using two prostate carcinoma cell lines, AT-3 and DT-3 cells, the present inventors then examined whether the reporter system could reflect cell type-specific splicing regulation. AT-3 cell is a mesenchymal-type cell that specifically expresses exon 9 isoform of endogenous FGFR2, and DT-3 cell is an epithelial-type cell that predominantly expresses exon 8 isoform [Yan G, et al., (1993) Mol Cell Biol 13: 4513-4522.]. When this reporter system was introduced into these two cell lines, AT-3 cell specifically expressed exon 9-RFP and DT-3 cell predominantly expressed exon 8-EGFP (FIG. 6B). The present inventors therefore could confirm that their reporter vector reflects cell type-specific regulation of endogenous FGFR2 splicing.
The present inventors next checked whether their reporter system could show tissue-specific regulation of FGFR2 splicing in vivo by generating transgenic mice from this reporter. A well-known change in the FGFR2 splicing isoforms occurs in mouse development stages from E14.5 to E16.5. In these stages, differentiation of future epithelial cells is induced and they start expressing exon 8 isoform of FGFR2 to receive morphogen signals, such as FGF-10, from mesenchymal cells [Finch P W, et al., (1995) Dev Dyn 203: 223-240.]. When the whole body of a transgenic embryo was examined at E14.5, a broad RFP signal was detected throughout the entire body, and a specific EGFP signal was detected as the whisker pattern and on the edges of limbs or the body (FIG. 6C). The present inventors further evaluated the detailed expression profile through examining series of sections from the transgenic embryos in the late development stage of E16.5 (FIG. 6D). An EGFP signal was detected specifically in cells on the surface of the skin and bulbs of hair follicles, where differentiated epithelial cells were located (FIG. 6D, shown with arrows). Also, the EGFP signal was detected at epithelial cells in the alveoli of the lung, in the esophagus and colon, at the thymus epithelia, and at the salivary gland ductal cells (FIG. 6D, arrows in the top and middle panels). A strong RFP signal was detected in the developing brain (hippocampus) and peripheral nervous system (trigeminal ganglia) (FIG. 6D, arrows in bottom panels). Expression patterns of EGFP were compatible with reported FGFR2 exon 8 expression patterns [Finch P W, et al., (1995) Dev Dyn 203: 223-240. Orr-Urtreger A, et al. (1993) Dev Biol 158: 475-486.], indicating that the present inventors' reporter system reflects endogenous splicing regulation of FGFR2 in vivo and the genomic fragment used in the vector contains the regulatory elements necessary for tissue-specific switching of mutually exclusive exons.
Example 4
Unbalanced Sequence of 3′ Splice Site is Essential for Mutually Exclusive Exon Selection
In the embryos of splicing reporter transgenic mouse, RFP was expressed almost throughout the entire body, and EGFP was specifically expressed in epithelial cells. This expression pattern suggest the possible regulatory mechanism that exon 9 was dominantly selected as “default” in reporter transgenic mouse, and epithelial-specific regulators might promote inclusion of exon 8. To test this hypothesis, the present inventors initially compared sequence of alternative exons including their 3′ and 5′ splice sites. The major difference identified between these two exons is that exon 8 has a weaker 3′ splice site and a polypyrimidine moiety that contains several mismatches from the consensus sequence (FIG. 7A, TGTTCTAG ca/SEQ ID NO: 40), whereas exon 9 has stronger 3′ splice site which has conserved consensus sequences (FIG. 7A, TTTTCTAG gc/SEQ ID NO: 41). There are no obvious differences in their 5′ splice sites (data not shown). To examine whether the unbalanced 3′ splice site is essential for “default” selection of exon 9 in non-epithelial cells, the present inventors introduced mutations in their 3′ splice sites and observed change in splicing regulations. The present inventors prepared two types of mutated vectors, one has the same stronger 3′ splice sites on both exon 8 and 9 (FIG. 7A, E8-S vector), and the other has the same weaker 3′ splice sites on both exon 8 and 9 (FIG. 7A, E9-W vector). These vectors were transfected into AT-3 and DT-3 cells, and the change of splicing regulation was examined by RT-PCR. When WT vector was introduced into AT-3 cell, it adopted almost 100% of the exon 9 form, whereas DT-3 cell adopted around 45% {34.5/(34.5+42.7) %} of exon 8 form among the single inclusion product (FIG. 7B, lane 1, 4), which was consistent with the expression pattern of fluorescence in FIG. 6B. The present inventors' splicing reporter was designed not to cause early premature termination codon in double-inclusion form to escape the nonsense-mediated decay (NMD) reaction [Chang Y F, et al., (2007) Annu Rev Biochem 76: 51-74]. The present inventors therefore could monitor all splicing products, including the double-inclusion and the double-skip forms. Strikingly, when E8-S vectors were transfected, AT-3 cell mostly expressed the double-inclusion form, meaning that two alternative exons were processed as constitutive exons (FIG. 7B, lane 2). This result indicates that the weaker 3′ splice site of exon 8 is critical for single exon selection of exon 9 from two mutually exclusive exons in non-epithelial AT-3 cell (FIG. 7B, lane 2). Also, this result indicated that exon 8 in AT-3 cell is not fully silenced by the hypothetical repressor(s), but merely ignored as a non-exon sequence in WT vector due to its weaker 3′ splice site, because the reported exonic and intronic splice silencers sequence around exon 8 were not mutated in these vectors [Carstens R P, et al., (2000) Mol Cell Biol 20: 7388-7400, Del Gatto F, Breathnach R (1995) Mol Cell Biol 15: 4825-4834, Wagner E J, et al. (2005) J Biol Chem 280: 14017-14027.]. When the 3′ splice site of exon 9 was weakened (E9-W vector), selection in DT-3 cell almost fully switched to exon 8 (FIG. 7B, lane 4 and 6), indicating that full repression of exon 9 might be important for exon 8 inclusion in epithelial DT-3 cell. Thus unbalanced 3′ splice sites are essential for the single exon choice from mutually exclusive exons and for their switching.
Example 5
Disruption of Exon 9 Causes Switching to Exon 8
Results in FIG. 7 showed that weaker 3′ splice sites of exon 8 is essential for the single exon selection of exon 9 in non-epithelial AT-3 cell. And epithelial DT-3 cell efficiently chose exon 8 form when 3′ splice site of exon 9 was weakened. These observations suggest a possibility that repression of exon 9 causes switching to exon 8. To examine this hypothesis, the present inventors introduced mutations on either or both of the 3′ and 5′ splice sites of exon 9 to destroy its splice site consensus sequence mimicking repression, and transfected these into AT-3 cell (FIG. 8A). When both splice sites of exon 9 were mutated (FIG. 8A, 3′&5′ ss Mut), AT-3 cell expressed the exon 8 form (22.9%) and the double-skip form (77.1%) (FIG. 8B, lane 4). These results indicated that blocking of exon 9, at least partially, promotes switching to exon 8 in AT-3 cell. Interestingly, mutation of the 3′ splice site (FIG. 8A, 3′ ss Mut) was just sufficient to cause this switching (FIG. 8B, lane 2), whereas mutation of the 5′ splice site (FIG. 8A, 5′ ss Mut) produced an aberrant splicing product of exon 9 using a cryptic 5′ splice site at ggGT in exon 9 (FIG. 8B, lane 3 indicated by arrowhead and scheme was illustrated on the right side). These results indicate that recognition of 3′ splice site is essential for exon 9 selection, suggesting the possibility that recognition of exon 9 is its 3′ splice site dependent. To test this hypothesis, the present inventors performed in vitro splicing assay to directly monitor the splice site recognition by U2 and U1 snRNA/snRNP binding (FIG. 8C). The 32P-labeled RNA probes for wild-type and mutated exon 9 containing the flanking introns (top panels of FIG. 8C) were crosslinked by UV irradiation after incubation with HeLa nuclear extract and separated by electrophoresis. HeLa cell was confirmed to have non-epithelial cell character as shown in FIG. 10A. The specificity of U2 or U1 binding was confirmed by addition of an oligonucleotide complementary to U2 or U1, and RNase H digestion [Konarska M M, Sharp P A (1986) Cell 46: 845-855, Sawa H, Shimura Y (1992) Genes Dev 6: 244-254.] (FIG. 8C). Under splicing conditions, binding and shift of U1 and U2 snRNAs were observed with the WT RNA probe, in which the U1 and U2 bands overlap (FIG. 8C, lane 1-4, indicated by arrow). Strikingly, probe possessing the 3′ splice site mutation resulted in a total loss of shifted band in both U1 and U2 (FIG. 8C, lane 5-8). These results showed that recognition of exon 9 totally depends on the binding of U2 snRNA to the 3′ splice site. To the contrary with the probe harboring with 5′ splice site mutation (5′ ss mutation), binding of both U1 and U2 was retained (FIG. 8C, lane 9-12), in good accordance with the results of RT-PCR shown in FIG. 8B, lane 3. These results suggest a possibility that binding of U2 snRNA supports the binding of U1 snRNA, so that much weaker cryptic 5′ splice sites in exon 9 was used in its 5′ ss mutation (FIG. 8B, lane 3 and FIG. 8C, lane 12 indicated by arrowheads with asterisk). These observations can explain why the selective use of exon 9 in non-epithelial cells depends on the relative strength of its 3′ splice site. A model shown in FIG. 12A, Non-Epithelial or Mesenchymal regulation could be proposed.
Example 6
Identification of Silencing Elements for Exon 9 Recognition
As shown in FIG. 12A, Non-Epithelial or Mesenchymal regulation, unbalanced 3′ splice sites are essential for single exon selection of exon 9 in non-epithelial cells and recognition of exon 9 is its 3′ splice site dependent. Also disruption of this 3′ splice site of exon 9 partially caused switching to exon 8. These results suggest the presence of silencer(s) for exon 9 to cause switching to exon 8 in epithelial cell. To test this hypothesis, the present inventors initially screened suppressive cis-elements located near the 3′ splice site of exon 9, and picked up two highly conserved sequences: the UGCAUG sequence and ISE/ISS-3 (intronic splicing enhancer/silencer-3) in intron 8, both of which have been reported as the silencing cis-elements for exon 9 [Baraniak A P, et al., (2006) Mol Cell Biol 26: 1209-1222, Hovhannisyan R H, Carstens R P (2005) Mol Cell Biol 25: 250-263.] (FIG. 9A). To examine whether these two cis-elements are essential for silencing exon 9, the present inventors introduced mutations in either or both UGCAUG and ISE/ISS-3 in their reporter, and transfected into epithelial DT-3 cell. First, the present inventors substituted UGCAUGCAUG (SEQ ID NO: 42) for UACGUACGUG (SEQ ID NO: 43) to disrupt the binding to the RNA-binding protein of Fox, which was reported as the repressor of exon 9 [Baraniak A P, et al., (2006) Mol Cell Biol 26: 1209-1222]. Then, the ratio of exon 8 selection in DT-3 cell fell by a half (44.7% to 21.9%, FIG. 9B, lane 2). Next, the present inventors deleted ISE/ISS-3, an 85-bp sequence containing several dinucleotide GU sequences. The deletion of ISE/ISS-3 reduced the ratio of exon 8 inclusion to one-fourth (44.7% to 12.4%, FIG. 9B, lane 3). When both of these elements were mutated, DT-3 cell could no longer choose exon 8, and all splicing products were the exon 9 form (FIG. 9B, lane 4). These results indicate that DT-3 cell use both of these cis-elements to select exon 8 presumably by silencing the exon 9 via its 3′ splice site.
Example 7
Regulatory Mechanism of Transacting Factors to Switch Exons
Results from FIG. 9 suggest that both UGCAUG and ISE/ISS-3 are necessary and sufficient for selecting exon 8. A previous study has shown that Fox2 promotes exon 8 inclusion through UGCAUG in intron 8 [Baraniak A P, et al., (2006) Mol Cell Biol 26: 1209-1222.]. Also, a recent study from cDNA library screening identified epithelial splicing regulatory protein ESRP1 and ESRP2, which mediate exon 8 inclusion through binding to ISE/ISS-3 [Warzecha C C, et al., (2009) Mol Cell 33: 591-601.]. The present inventors therefore examined whether Fox1, Fox2, ESRP1, and ESRP2 promote switching from exon 9 to exon 8. First, the present inventors examined and compared the expression levels of these RNA-binding proteins between AT-3 and DT-3 cells by RT-PCR. Fox2 was expressed in both cell lines at similar levels, whereas expression of Fox1 was undetectable (FIG. 9C), and both ESRP1 and ESRP2 were specifically expressed in epithelial type DT-3 cell (FIG. 9C). Considering the observation that both UGCAUG and ISE/ISS-3 are essential cis-elements for selecting exon 8 (FIG. 9B), broadly expressed Fox2 might cooperates with epithelial-specific ESRP1 and ESRP2 for exon 8 inclusion. To test this hypothesis, the present inventors transfected their FGFR2 splicing reporter with Fox1, Fox2, ESRP1, or ESRP2, or combinations of these into HeLa cell, which has non-epithelial cell character (FIG. 10A, lane 1). When Fox1 or Fox2 was introduced into HeLa cell, selection of exon 8 increased in a dose-depend manner and reached 10% (FIG. 10A, lanes 2-4) or 40% (FIG. 10A, lanes 5-7), respectively. When ESRP1 or ESRP2 was introduced, selection of exon 8 also increased in a dose-depend manner, and reached over 90% (FIG. 10A, lanes 8-10 and 15-17, respectively). As HeLa cell expresses endogenous Fox2 in a similar manner to AT-3 cell (FIG. 10A, lane 1), the present inventors introduced ESRP1 or ESRP2 under the Fox2 knockdown condition, and evaluated the cooperative effects of ESRP1 or ESRP2 with endogenous Fox2. The knockdown efficiency of Fox2 was more than 80% at the mRNA level (average 81.2%). Knockdown of endogenous Fox2 decreased the ratio of exon 8 selection promoted by ESRP1 or ESRP2, with a maximum reduction of 24% (35.1-11.0% FIG. 10A, lane 8 versus lane 11) or 23% (55.9-32.6%, lane 15 versus lane 18), respectively. When both Fox2 and ESRP1 were transfected, the ratios of exon 8 inclusion were similar to those obtained with a single transfection of ESRP1 (FIG. 10A, lanes 21-24). These results indicate that introduced ESRPs promote exon 8 inclusion with endogenous Fox2, suggesting that Fox2 and ESRPs cooperatively act together for exon 8 inclusion. These results were also confirmed by means of the fluorescence from the splicing reporter co-transfected with Fox1, Fox2, ESRP1, or ESRP2, or both Fox2 and ESRP1 (FIG. 10B). Overexpression of ESRP1 or ESRP2 changed the color from red to green, but Fox1 or Fox2 alone had a smaller effect on the color change. Co-transfection of Fox2 and ESRP1 gave the maximum effect on the color switching which reflected the switching of proteins coded by the mutually exclusive exons. The present inventors then tested whether exon switching by Fox2 and ESRP1 depends on UGCAUG and ISE/ISS-3 in intron 8. The present inventors performed overexpression study of Fox2 and/or ESRP1 on reporter vectors mutated on either UGCAUG, ISE/ISS-3, or both of them, and confirmed that exon switching caused by Fox and ESRPs depends on UGCAUG and ISE/ISS-3, respectively (FIG. 13). These results showed that Fox and ESRP cooperatively promote switching from exon 8 to 9 through the cis-elements of UGCAUG and ISE/ISS-3 located near exon 9.
Next, the present inventors tested whether Fox and/or ESRP cause switching from exon 9 to exon 8 through interruption of exon 9 recognition. The present inventors examined this under the in vitro splicing conditions using 32P-labeled RNA probe of exon 9 with introns containing UGCAUG and ISE/ISS-3 sites (FIG. 10C, top panel) and the exon 8 RNA probe of same stretches. When the exon 9 probe was crosslinked by UV irradiation after incubation with HeLa nuclear extract and separated by electrophoresis, shifted band by closslinking U1 and U2 was observed as overlap (FIG. 10C, lane 2, indicated by arrow), as same as FIG. 8C. However, in the exon 8 probe, only shifted band by U1 was observed (FIG. 10C, lane 8, indicated by arrow, and data not shown for RNaseH digestion), presumably due to its weaker 3′ splice site. When recombinant Fox2 or ESRP1 protein was added with the exon 9 probe, shifted binds by U1 and U2 snRNA were decreased (FIG. 10C, lanes 4 and 5, respectively), and almost disappeared by addition of both Fox2 and ESRP1 proteins (FIG. 10C, lane 6). However, suppressive or activating effect of Fox2 or/and ESRP1 was not obvious with exon 8 probe (FIG. 10C, lanes 9-12). These data indicate that both Fox and ESRP interrupt exon 9 recognition in vitro. Combining results from FIG. 10 and FIG. 13 indicate that Fox and ESRP disrupt exon 9 recognition from its 3′ splice site through UGCAUG and ISE/ISS-3, and promoted switching to exon 8. A model shown in FIG. 12A, Epithelial regulation could be proposed.
Example 8
Expression Profile of Fox and ESRP Coincide with Exon8-EGFP In Vivo
In vitro study showed that disruption of exon 9 recognition from its 3′ splice site by Fox and ESRP through UGCAUG and ISE/ISS-3 promoted switching to exon 8. Remaining question is whether the expression of Fox and ESRP coincides with the expression of exon 8 form in tissue-specific manner during development in vivo. The present inventors examined the expression profiles of Fox1, Fox2, ESRP1, and ESRP2 by in situ hybridization using the serial sections from their reporter transgenic mice embryos at E16.5 (FIG. 11). As the present inventors have already shown in FIG. 6D, the exon 8-EGFP expression was on left panels (as indicated by white arrows). In the in situ hybridization performed with adjacent sections, Fox1 mRNA was detected broadly throughout whole embryos at this stage, and its expression was overlapped with exon 8-EGFP signals localized in the epithelial tissues (indicated by black arrows). However, Fox2 was not detected in tissues where the EGFP signal was observed, whereas strong signal of Fox2 was detected in neuronal tissues and muscle in the same sections (data not shown). The expression of ESRP1 and ESRP2 was specifically detected in epithelial tissues (indicated by black arrows) and these expression almost completely overlapped with exon 8-EGFP signals during developing stage. These observations in vivo support an epithelial regulation model in FIG. 12A in which tissue specific factor ESRPs act together with generally expressed Fox family to promote exon 8 inclusion.
Example 9
A Method for Testing Whether or not a Compound to be Tested Affects an Alternative Splicing of a Specific Gene in a Mammalian Multicelluar Organism
Strategy of a method for testing whether or not a compound to be tested affects an alternative splicing of a specific gene in a mammalian multicelluar organism is shown in FIG. 14.
The reporter proteins used for the method are GFP and FRP. Results of the method are shown in Tables 4-1 to 4-22. These results demonstrate that compounds that affect an alternative splicing of a specific gene have been actually isolated by the method of the present invention.


















TABLE 4-1















Ctrl











final conc.
Number
number
GFP/RFP


#
Location
Cat #
Name
CAS
MW
Cone
ID #
(μM, 50 ng/mL)
of cells
of cells
(Ctrl = 1)


























272
4-D3
GR-319
Doxorubicin HCl
25316-40-9
543.5
2 mg/ml
66
9.199085655 
18
5290
18.69526351


271
4-D2
GR-318
Daunorubicin HCl
23541-50-6
527.5
2 mg/ml
191
9.4780822 
3
5290
11.83377412


263
4-C4
GR-301
Camptothecin
7689-03-4
348.4
2 mg/ml
288
14.35290557
385
5290
5.440752087


238
3-H9
EI-206
Auranofin
34031-32-8
678.5
2 mg/ml
758
7.369268658
1
4352
4.915173713


172
3-B3
DL-548
Idarubicin 
58957-92-9
497.5
2 mg/ml
253
10.05012217
34
4352
4.863921716


408
6-A9
DL-370
Cytarabine 
147-94-4
243.2
2 mg/ml
147
20.55743035
48
4303
4.588269745


267
4-C8
GR-311
Mitomycin c
50-07-7
334.3
2 mg/ml
387
14.95508196
319
5290
3.767284647


314
4-H5
DL-215
Gemcitabine HCl
95058-81-4
263.2
2 mg/ml
392
18.9967786
156
5290
3.517172864


407
6-A8
DL-369
Cyclocytidine HCl
10212-25-6
225.2
2 mg/ml
52
22.20192276 
73
4303
3.416714729


441
6-E2
DL-399
5-fluorouracil
51-21-8
130.1
2 mg/ml
42
38.43814737 
433
4303
3.160858079


531
7-F2
DL-488
Quinacrine 2HCl 
83-89-6
400.0
2 mg/ml
604
12.50099852 
5
4940
3.152823983





dihydrate










270
4-C11
GR-316
10-hydroxycamptothecin
64439-81-2
364.4
2 mg/ml
13
13.72265719 
541
5290
3.113545343


445
6-E6
DL-403
Floxuridine
50-91-9
246.2
2 mg/ml
152
20.30895444 
142
4303
3.000014372


591
8-D2
AC-1053
Harmine
442-51-3
212.3
2 mg/ml
127
23.55674979 
537
4361
2.851610014


484
7-A5
DL-442
Melphalan
148-82-3
305.2
2 mg/ml
247
16.38234506 
483
4940
2.82837027


323
5-A4
DL-103
Clofarabine
123318-82-1
303.7
2 mg/ml
4
16.46457661 
19
4930
2.692957739


492
7-B3
DL-450
Mitoxantrone 2HCl
65271-80-9
444.5
2 mg/ml
422
11.24880498 
31
4940
2.65051939


490
7-A11
DL-448
Miconazole
22916-47-8
416.1
2 mg/ml
309
12.01527151 
808
4940
2.531917074


493
7-B4
T-104
Taxol
33069-62-4
853.9
2 mg/ml
671
5.855288969 
719
4940
2.454988842


617
8-F8
PR-117
Artesunate
88495-63-0
384.4
2 mg/ml
136
13.00626192 
246
4361
2.378199382


318
4-H9
DL-303
Oxaliplatin
61825-94-3
397.3
2 mg/ml
524
12.58489202 
825
5290
2.271403553


569
8-A10
DL-518
Topotecan
123948-87-8
421.5
2 mg/ml
698
11.86361043 
259
4361
2.243826843


398
5-H9
DL-360
Clomiphene citrate
50-41-9
406.0
2 mg/ml
467
12.31611547 
95
4930
2.163748878


264
4-C5
GR-305
Plicamycin
18378-89-7
1085.2
2 mg/ml
570
4.607568252 
1070
5290
2.050967296


376
5-F7
DL-338
Bifonazole
60628-96-8
310.4
2 mg/ml
200
16.10813533 
921
4930
1.99842226


166
3-A7
AW8655
Aclarubicin
57576-44-0
811.9
2 mg/ml
5
6.1584818C4 
10
4352
1.998350103









Table 4-2 is a continuation of Table 4-1.


















TABLE 4-2







321
5-A2
DL-545
Mycophenolate 
128794-94-5
433.5 
2 mg/ml
11
11.5338652 
500
4930
1.989012913





mofetil










147 
2-G8
DL-208
Sertaconazole
99592-32-2
437.8
2 mg/ml
657
11.42130487 
1863
5627
1.988773841


412 
6-B3
DL-154
Desloratadine
100643-71-8
310.8
2 mg/ml
291
16.08597995 
231
4303
1.94912201


466
6-G7
DL-424
Imiquimod
99011-02-6
240.3
2 mg/ml
306
20.80642196 
1944
4303
1.902330123


382 
5-G3
DL-344
Carboplatin
41575-94-4
337.2
2 mg/ml
337
14.82789324 
1373
4930
1.874549862


133
2-F4
DL-250
Docetaxil
114977-28-5
807.9
2 mg/ml
84
6.188883537 
1194
5627
1.862180326


565 
8-A6
DL-515
Tioconazole
65899-73-2
387.7
2 mg/ml
688
12.8959771 
850
4361
1.840788999


52 
1-F3
AC-185
Dobutamine HCl
49745-95-1
301.4
2 mg/ml
429
16.58986059 
2991
5531
1.824866427


464 
6-G5
DL-422
Idoxuridine
54-42-2
354.1
2 mg/ml
549
14.12019255 
1924
4303
1.803725044


328
5-A9
DL-156
Lomustine
13010-47-4
233.7
2 mg/ml
375
21.39497185 
1241
4930
1.797894151


582 
8-C3
DL-530
Vidarabine
5536-17-4
267.2
2 mg/ml
726
18.70933923 
1482
4361
1.71874095


475 
6-H6
DL-433
Leflunomide
75706-12-6
270.2
2 mg/ml
176
18.50392577 
1963
4303
1.707457571


389 
5-G10
DL-351
Chlorambucil
305-03-3
304.2
2 mg/ml
330
16.43552673 
841
4930
1.661670317


266 
4-C7
GR-307
Etoposide
33419-42-0
588.6
2 mg/ml
367
8.495107735 
055
5290
1.649456036


612 
8-F3
PD-195
Zardaverine
101975-10-4
268.2 
2 mg/ml
738
18.64128171 
2744
4361
1.621688757


195 
3-D6
CA-210
Nifedipine
21829-25-4
346.3 
2 mg/ml
573
14.43656359 
156
4352
1.6143365


85 
2-AG
AC-729
Isoproterenol HCl (rac)
51-30-9
211.3
2 mg/ml
23
23.66717813 
3953
5627
1.61359971


586 
8-C7
NH-101
Astemizole
68844-77-9
458.6
2 mg/ml
137
10.90313098 
38
4361
1.594859469


110 
2-C11
AC-810
Norepinephrine-(+)-
50-40-1
319.2
2 mg/ml
741
15.66069709 
4210
5627
1.593775287





tartrate l (−)










202 
3-E3
CA-236
Felodipine
72509-76-3
384.2
2 mg/ml
174
13.01194082 
116
4352
1.579410212


570 
8-A11
DL-519
Toremifene
89778-26-7
406.0
2 mg/ml
699
12.31611547 
250
4361
1.574305192


553 
7-H4
DL-507
Sulfadoxine
2447-57-6
310.3
2 mg/ml
660
16.11168465 
2876
4940
1.545298551


108 
2-C9
AC-808
Epinephrine-(+)-
51-40-1
169.2
2 mg/ml
159
29.55401164 
4057
5627
1.528497942





tartrate l (−)










416 
6-B7
DL-377
Diethylstilbestrol
56-53-1
268.4
2 mg/ml
360
18.63176515 
542
4303
1.524990903


370 
5-E11
DL-332
Atracurium besylate
64228-81-5
929.2
2 mg/ml
145
5.381140881 
3056
4930
1.520793417


8 
1-A9
A-249
Mycophenolic acid
24280-93-1
320.3
2 mg/ml
481
15.60815539 
1367
5531
1.512459974


273 
4-D4
GR336
Tanshinone iia
568-72-9
294.4
2 mg/ml
668
16.98637805 
2210
5290
1.504678809


469 
6-G10
DL-427
Itraconazole
84625-61-6
705.7
2 mg/ml
29
7.085662585 
344
4303
1.5046657


485 
7-A6
DL-443
Methyldopa
555-30-6
211.2 
2 mg/ml
579
23.67206688 
3217
4940
1.501882352









Table 4-3 is a continuation of Table 4-2.


















TABLE 4-3







287
4-E8
DL-297
Bortezomib
179324-69-7
384.2
2 mg/ml
218
13.01248027
74
5290
1.496452459


435
6-D6
DL-393
Fenbendazole
43210-67-9
299.4
2 mg/ml
10
16.70264639
278
4303
1.49457566


158
2-H9
AP-302
Bleomycin sulfate
9041-93-4
1415.6
2 mg/ml
210
3.532131015
2702
5627
1.477092334


507
7-C8
DL-464
Oxiconazole nitrate
64211-46-7
429.1
2 mg/ml
529
11.65132316
2732
4940
1.459520403


418
6-B9
DL-379
Disulfiram
97-77-8
296.5
2 mg/ml
391
16.86111466
31
4303
1.442217134


549
7-G10
DL-149
Sparfloxacin
110871-86-8
392.4
2 mg/ml
624
12.74180798
3029
4940
1.44013786


522
7-E3
DL-479
Prednisolone
50-24-8
360.5
2 mg/ml
555
13.87138359
2900
4940
1.433017135


614
8-F5
PG-051
Misoprostol
59122-46-2
382.5
2 mg/ml
405
13.07035227
3052
4361
1.431648768


559
7-H10
DL-512
Tenoxicam
59804-37-4
337.4
2 mg/ml
676
14.82015798
2916
4940
1.429499751


530
7-E11
DL-487
Pyrantel pamoate
22204-24-6
594.7
2 mg/ml
602
8.407713573
2985
4940
1.406116379


635
8-H6
DL-225
Phenylpropanolamine
14838-15-4
151.2
2 mg/ml
542
33.066583
2551
4361
1.403544143


555
7-H6
DL-509
Sulfadimethoxine
122-11-2
310.3
2 mg/ml
659
16.11168465
3092
4940
1.399151769


225
3-G6
EI-125
Capsaicin
404-86-4
305.4
2 mg/ml
310
16.37085674
1918
4352
1.397969148


613
8-F4
PG-006 
Alprostadil
745-65-3
354.5
2 mg/ml
74
14.10473124
3505
4361
1.396557197




PGE1











345
5-C6
DL-175
Tulobuterol
41570-61-0
227.7
2 mg/ml
717
21.95521172
3489
4930
1.391996805


342
5-C3
DL-148
Ricobendazole
54029-12-8
281.3
2 mg/ml
621
17.7723655
1381
4930
1.386396885


510
7-C11
DL-467
Oxibendazole
20559-55-1
249.3
2 mg/ml
528
20.05843825
786
4940
1.381170954


521
7-E2
DL-478
Pranoprofen
52549-17-4
255.3
2 mg/ml
409
19.58666189
3050
4040
1.364655602


540
7-F11
ST-405
Propranolol
3506-09-0
259.4
2 mg/ml
568
19.27887224
2641
4940
1.362604454


170
3-A11
DL-270
Ibudilast
50847-11-5
230.3
2 mg/ml
353
21.70964685
3018
4352
1.36174848


94
2-B5
AC-748
Apomorphine r (−)
314-19-2
267.3
2 mg/ml
495
18.70343733
3372
5627
1.356935081


560
7-H11
DL-513
Terazosin HCl
63590-64-7
387.4
2 mg/ml
677
12.90515076
3112
4940
1.355776526


509
7-C10
DL-466
Oxfendazole
53716-50-0
315.4
2 mg/ml
527
15.85523988
1712
4940
1.351521473


486
7-A7
DL-444
Methylprednisolone
83-43-2
374.5
2 mg/ml
202
13.35179798
3084
4940
1.347926167


286
4-E7
PR-118
Pentamidine
100-33-4
340.4
2 mg/ml
408
14.68736202
2694
5290
1.34452932


520
7-D11
DL-477
Pravastatin lactone
NA
406.5
2 mg/ml
591
12.29940198
2968
4940
1.341716934


544
7-G5
DL-499
Satafloxacin HCl
91296-87-6
385.4
2 mg/ml
647
12.97442308
3105
4940
1.340643527


536
7-F7
DL-147
Rebamipide
90098-04-7
370.8
2 mg/ml
612
13.48452543
2922
4940
1.337638677


511
7-D2
DL-468
Pantoprazole
102625-70-7
383.4
2 mg/ml
388
13.04201132
3112
4940
1.332777083









Table 4-4 is a continuation of Table 4-3.


















TABLE 4-4







498
7-B9
DL-456
Nefazodone
83366-66-9
470.0
2 mg/ml 
72
10.63786579 
2966
4940 
1.321266691


245
4-A6
EI-265
Carbidopa
28860-95-9
226.2
2 mg/ml
336
22.10100264 
3470
5290 
1.316427838


48
1-E9
AC-175
Procaterol hcl
62929-91-3
290.4
2 mg/ml 
561
17.21968607 
4151
5531 
1.312595792


630
8-G11
DL-104
Fulvestrant
129453-61-8
606.8
2 mg/ml
272
8.240142947 
2156
4361 
1.312106951


399
5-H10
DL-361
Clopidol
2971-90-6
192.0
2 mg/ml 
476
26.03543715 
3230
4930 
1.310007746


10
1-A11
A-275
Rapamycin
53123-88-9
914.2
2 mg/ml
608
5.469279614 
2046
5531 
1.309221229


302
4-G3
DL-222
Alfacalcidol
41294-56-8
400.7
2 mg/ml 
63
12.47970395 
861
5290 
1.301315546


624
8-G5
DL-532
Amprenavir
161814-49-9
505.6
2 mg/ml
101
9.888493393 
2564
4361 
1.2958728


49
1-E10
AC-176
Salbutamol sulfate
51022-70-9
239.3
2 mg/ml 
643
20.89277153 
4053
5531
1.29533558


355
5-D6
DL-317
3′-azido-3′-
30516-87-1
267.2
2 mg/ml
16
18.70933923 
2356
4930 
1.294443542





deoxythymidine










607
8-E8
PD-175
Rolipram
61413-54-5
275.4
2 mg/ml 
635
18.15866292 
2878
4361 
1.29431941


558
7-H9
DL-511
Telmisartan
144701-48-4
514.6
2 mg/ml
673
9.71566806 
3042
4940 
1.290679894


609
8-E10
PD180
Trequinsin
79855-88-2
405.5
2 mg/ml 
706
12.33042308 
3009
4361 
1.28875769


500
7-B11
DL-458
Norethindrone
68-22-4
298.4
2 mg/ml
163
16.75440277 
3205
4940 
1.288435074


381
5-G2
DL-343
Carbadox
6804-07-5
262.2
2 mg/ml 
317
19.06746737 
2293
4930 
1.280857769


636
8-H7
DL-234
Succinylcholine
306-40-1
290.4
2 mg/ml
654
17.21726327 
2727
4361 
1.275426477


571
8-B2
DL-520
Tolfenamic acid 
13710-19-5
261.7
2 mg/ml 
695
19.10509883 
2697
4361 
1.274462393


34
1-D5
AC-146
Prazosin HCl 
19216-56-9
383.4
2 mg/ml
554
13.0408592 
2109
5531 
1.27325226


392
5-H3
T-115
Thaldomide 
50-35-1
258.2
2 mg/ml 
681
19.3621601 
2622
4930
1.27114327


568
8-A9
DL-188
Tosufloxacin 
100490-36-6
404.4
2 mg/ml
700
12.36547599 
2590
4361 
1.265839944


159
2-H10
AR-100
Bopindolol malonate 
62658-64-4
380.5
2 mg/ml 
215
13.14090804 
3625
5627 
1.26531707


298
4-F9
DL-299
Celecoxib 
169590-42-5
331.4
2 mg/ml
328
13.11031018 
3060
5290 
1.262654116


461
6-G2
DL-419
Hydrocortisone
50-23-7
362.5
2 mg/ml 
189
13.79423553 
3105
4303 
1.261714165


259
4-B10
GR-100
Retinoic acid
302-73-4
300.4
2 mg/ml
617
16.64198328 
2697
5290 
1.260341848


532
7-F3
DL-489
Quinapril HCl
82586-55-8
438.5
2 mg/ml 
606
11.401774 
3149
4940
1.258641379


625
8-G6
DL-533
Aprepitant
170729-80-3
534.4
2 mg/ml
118
9.35562882 
2165
4361 
1.253657712


502
7-C3
DL-460
Nystatin
1400-61-9
926.1
2 mg/ml 
198
5.398878307 
3293
4940
1.252683637


537
7-F8
DL-492
Ribavirin 
36791-04-5
244.2
2 mg/ml
619
20.47429608 
3140
4940 
1.249983388


390
5-G11
DL-352
Chlorpheniramine 
113-92-8
274.8
2 mg/ml 
365
18.19530057 
3724
4930 
1.2493157





maleate

















Table 4-5 is a continuation of Table 4-4.


















TABLE 4-5







334
5-B5
DL-309
Octreotide
83150-76-9
1019.3
2 mg/ml
206
4.905513097
3742 
4930
1.242156182


514
7-D5 
DL-471
Pefloxacine mesylate
149676-40-4
333.4
2 mg/ml
535
14.99854589
3646 
4940
1.236573794


86
2-A7
AC-734
Acetylcholine Cl
60-31-1
146.2
2 mg/ml
50 
34.19713706
3490
5627
1.235291184


146
2-G7
DL-262
Atovaquone
95233-18-4
366.8
2 mg/ml
140
13.62962577
3754 
5627
1.229980859


470
6-G11
EI-288
Ketoprofen (s)
2216-88-5
254.3
2 mg/ml
95 
19.66273069
2780 
4303
1.229222044


119
2-D10
DL-124
Vitamin a (acetate) 
68-26-8
328.5
2 mg/ml
732
15.22074
3527 
5627
1.228127881


296
4-F7
DL-298
Bexarotene
153559-49-0
348.5
2 mg/ml
181
14.34763211
3070 
5290
1.224129173


425
6-C6
DL-384
Enrofloxacin
93106-60-6
359.4
2 mg/ml
135 
13.9119251
3025
4303
1.223123525


357
5-D8
PR-123
Alendronate
121268-17-5
248.1
2 mg/ml
62
20.15394876
3185
4930
1.220700948


92
2-B3
AC-746
Tubocurarine Cl (+)
57-94-3
609.7
2 mg/ml
716
8.200093835
3433 
5627
1.219635948


451
6-F2
DL-409
Ftorafur
17902-23-7
200.2
2 mg/ml
268
24.978652
2962 
4303
1.218453142


127
2-E8
AC-986
Isoniazid
54-85-3
137.1
2 mg/ml
15 
36.45851069
3759
5627
1.21783323


623
8-G4
S-650
Tamoxifen citrate
54965-24-1
371.5
2 mg/ml
666
13.45796739
2282 
4361
1.215330405


447
6-E8
DL-405
Flubendazole
31430-15-6
313.3
2 mg/ml
153
15.9596163
409 
4303
1.212383568


61
1-G2
AC-221
Telenzepine 2HCl
147416-96-4
370.5
2 mg/ml
672
13.49612212
3723 
5531
1.210899438


144
2-G5
DL-260
Ractopamine
97825-25-7
301.4
2 mg/ml
607
16.58986059
3997 
5627
1.209935549


634
8-H5
DL-146
Phloridzin
60-81-1
436.4
2 mg/ml
39
11.45686845
2768 
4361
1.206731926


165
3-A6
DL-223
Cerivastatin
145599-86-6
459.6
2 mg/ml
329
10.8799016
108 
4352
1.205442874


118
2-D9
AC-858
Naltriben mesylate
111555-58-9
415.5
2 mg/ml
489
12.03388405
3827
5627
1.203653932


99
2-B10
AC-757
Raclopride l-tartrate s(−)
84225-95-6
347.2
2 mg/ml
603 
14.39908652
3519 
5627
1.203305219


140
2-F11
DL-256
Lacidipine
103890-78-4
455.6
2 mg/ml
130
10.97559881
3439 
5627
1.20137176


128
2-E9
AC-993
Ticlopidine HCl
53885-35-1
263.8
2 mg/ml
685 
18.9543722
3369
5627
1.201082388


602
8-E3
PD-125
Cilostamide 
68550-75-4
342.4
2 mg/ml
373 
14.6010204
2851 
4361
1.199961281


528
7-E9
DL-485
Primaquine phosphate
63-45-6
259.4
2 mg/ml
592 
19.27866039
3270 
4940
1.198460584


506
7-C7
DL-463
Oxcarbazepine
28721-07-5
252.3
2 mg/ml
526
19.81963419
3556
4940
1.198397854


160
2-H11
AR-102
Guanfacine HCl
29520-14-7
282.6
2 mg/ml
8 
17.69544755
3849 
5627
1.197498136


261
4-C2
GR-211
Bezafibrate
41859-67-0
361.8
2 mg/ml
183
13.81869949
3845 
5290
1.195438873


556
7-H7
DL-510
Sulfasalazine
599-79-1
398.4
2 mg/ml
661 
12.55019824
3292
4940
1.192301974


482
7-A3
DL-440
Medroxyprogesterone
98-92-0
122.1
2 mg/ml
144 
40.94081334
3282 
4940
1.191977459





17-acetate










380
5-F11
DL-342
Canrenone
976-71-6
340.5
2 mg/ml
299
14.68572576
3703
4930
1.190307786









Table 4-6 is a continuation of Table 4-5.


















TABLE 4-6







439
6-D10
DL-397
Fenofibrate
49562-28-9
360.8
2 mg/ml
35
13.85651967
2923
4303 
1.189725982


547
7-G8
DL-502
Secnidazole
3366-95-8
185.2
2 mg/ml
653
27.00016989 
3233
4940
1.188643076


444
6-E5
DL-402
Florfenicol
73231-34-2
358.2
2 mg/ml
151
13.95797778
2867
4303
1.187670804


638
8-H9
DL-540
L-thyroxine [(3-[4-(4-
51-48-9
776.9
2 mg/ml
747
6.436026756
2477
4361
1.183555403





hydroxy-3,5-













diiodophenoxy)-3,5-













diiodophenyl]-l-alanine]










481
7-A2
DL-439
Mebendazol
31431-39-7
295.3
2 mg/ml
75
16.93191585
572
4940
1.176735236


254
4-B5
G-233
Mevastatin
73573-88-3
390.5
2 mg/ml
297
12.80329633
1633
5290
1.176328614


93
2-B4
AC-747
Butaclamol (+)
55528-07-9
361.5
2 mg/ml
261
13.83003747
3384
5627
1.175993589


515
7-D6
DL-472
Penciclovir
39809-25-1
253.3
2 mg/ml
536
19.74234269
3564
4940
1.174629247


268
4-C9
GR-312
Puromycin 2HCl
58-58-2
471.5
2 mg/ml
600
10.60399665
42
5290
1.173417452


359
5-D10
DL321
Albendazole
54965-21-8
265.3
2 mg/ml
61
18.84401396
504
4930
1.166836827


142
2-G3
G-226
Lovastatin
75330-75-5
404.6
2 mg/ml
582
12.35936525
1348
5627
1.16576373


134
2-F5
DL-251
Olopatadine
113806-05-6
337.4
2 mg/ml
246
14.81822366
3388
5627
1.164020602


234
3-H5
EI-166
Debrisoquin sulfate
581-88-4
175.2
2 mg/ml
214
28.53307848
3765
4352
1.162466552


535
7-F6
DL-491
Ramipril
87333-19-5
416.5
2 mg/ml
611
12.00417102
3358
4940
1.16242813


533
7-F4
DL-159
Racecadotril
81110-73-8
385.5
2 mg/ml
597
12.97064774
3502
4940
1.160386252


303
4-G4
DL-151
Anethole-trithione 
532-11-6
240.4
2 mg/ml
110
20.80155376
2915
5290
1.158201623





(anetholtrithion)










603
8-E4
PD-130
Etazolate
51022-77-6
289.3
2 mg/ml
303
17.28071797
2866
4361
1.157947621


562
8-A3
DL-169
Tenatoprazole
113712-98-4
346.4
2 mg/ml
675
14.43372763
2969
4361
1.157672912


518
7-D9
DL-475
Phenylbutazone
50-33-9
308.4
2 mg/ml
541
16.21358085
3490
4940
1.15747637


554
7-H5
DL-508
Sulfadiazine
68-35-9
250.3
2 mg/ml
658
19.9775612
3242
4940
1.157101921


443
6-E4
DL-401
Fleroxacin
79660-72-3
369.3
2 mg/ml
121
13.53742316
3292
4303
1.155032763


405
6-A6
DL-367
Cyclophosphamide 
6055-19-2
261.1
2 mg/ml
54
19.15051385
3065
4303
1.153475869





monohydrate










616
8-F7
PI-153
Cilastatin
82009-34-5
358.5
2 mg/ml
371
13.94855694
2885
4361
1.15300352


236
3-H7
EI-168
Bumetanide
28395-03-1
364.4
2 mg/ml
27
13.72030634
2977
4352
1.151305133


495
7-B6
DL-453
Nadifloxacin
124858-35-1
360.4
2 mg/ml
484
13.873912
3397
4940
1.150950193


152
2-H3
DL-200
Dorzolamide
120279-95-0
324.4
2 mg/ml
270
15.41107406
3803
5627
1.149226768









Table 4-7 is a continuation of Table 4-6.


















TABLE 4-7







611
8-F2
PD-190
Zaprinast
37762-06-4
271.3
2 mg/ml
737
18.43108283
2941
4361 
1.147348515 


529
7-E10
DL-486
Praziquantel
55268-74-1
312.4
2 mg/ml
553
16.00433628
2885
4940
1.145953739


575
8-B6
DL-524
Triamcinolone
124-94-7
394.4
2 mg/ml
707
12.67606612
2580
4361
1.143977924


377
5-F8
DL-339
Bisacodyl
603-50-9
361.4
2 mg/ml
207
13.83504635
3324
4930
1.143483143


422
6-C3
DL-381
Doxycycline HCl
24390-14-5
444.4
2 mg/ml
94
11.24998138
3267
4303
1.142311586


111
2-D2
DL-557
Quetiapine fumarate
111974-72-2
883.1
2 mg/ml
751
5.66183012
3666
5627
1.139672188


513
7-D4
DL-470
Pazufloxacin
127045-41-4
318.3
2 mg/ml
393
15.70808842
3492
4940
1.136211888


517
7-D8
DL-474
Pencillin v 
132-98-9
349.4
2 mg/ml
537
14.31072575
3442
4940
1.13425101





potassium










457
6-F8
DL-415
Gliclazide
21187-98-4
323.4
2 mg/ml
433
15.4599209
3316
4303
1.133895534


329
5-A10
DL-543
Iloprost
78919-13-8
360.5
2 mg/ml
359
13.86970478
3838
4930
1.132367656


525
7-E6
DL-482
Prothionamide
14222-60-7
180.3
2 mg/ml
598
27.73565195
5562
4940
1.132043112


69
1-G10
AC-277
Mepyramine maleate
59-33-6
285.4
2 mg/ml
452
17.51974019
3548
5531
1.131255358


579
8-B10
DL-527
Valaciclovir
124832-27-5
324.3
2 mg/ml
719
15.41581937
2836
4361
1.130813555


459
6-F10
DL-417
Guaifenesin
93-14-1
198.2
2 mg/ml
51
25.2244115
3239
4303
1.129530166


620
8-F11
S-510
Mifepristone
84371-65-3
429.6
2 mg/ml
316
11.63852239
1878
4361
1.12949478


534
7-F5
DL-490
Ranolazine 2HCl
95635-56-6
427.5
2 mg/ml
601
11.69458488
3509
4940
1.127775031


76
1-H7
DL-560
zolmitriptan
139264-17-8
303.4
2 mg/ml
461
16.4794763
3765
5531
1.127507134


476
6-H7
DL-434
Lisinopril
83915-83-7
405.5
2 mg/ml
302
12.33050731
3244
4303
1.127147071


197
3-D8
CA-212
Nitrendipine
39562-70-4
360.4
2 mg/ml
508
13.8746327
2606
4352
1.126633527


185
3-C6
PG-008
Dinoprost
551-11-1
354.5
2 mg/ml
141
14.10473124
3656
4352
1.125552615


255
4-B6
G-244
Simvastatin
79902-63-9
418.6
2 mg/ml
638
11.94518755
933
5290
1.124603165


541
7-G2
DL-496
Roxatidine acetate HCl
93793-83-0
348.4
2 mg/ml
637
14.34942532
3592
4940
1.123767026


332
5-B3
DL-167
Meropenem
96036-03-2
383.5
2 mg/ml
458
13.03883303
3402
4930
1.119846063


347
5-C8
DL-220
Vindesine
53643-48-4
753.9
2 mg/ml
729
6.631765014
653
4930
1.117956962


206
3-E7
DL-218
Denbufylline
57076-71-8
320.4
2 mg/ml
283
15.60575226
3308
4352
1.117337902


289
4-E10
DL-186
Fentiazac
18046-21-4
329.8
2 mg/ml
107
15.16034996
2880
5290
1.117182769


368
5-E9
NP-016
Artemisinin
63968-64-9
282.3
2 mg/ml
134
17.70917072
2662
4930
1.116460589


639
8-H10
DL-136
Cyproheptadine
129-03-3
287.4
2 mg/ml
119
17.39685803
2204
4361
1.115155722


115
2-D6
AC-834
Mesulergine HCl
64795-35-3
362.5
2 mg/ml
473
13.79319781
3333
5627
1.11434003


365
5-E6
DL-327
Ampicillin trihydrate
7177-48-2
349.4
2 mg/ml
100
14.30977392
3186
4930
1.113993115









Table 4-8 is a continuation of Table 4-7.


















TABLE 4-8







631
8-H2
DL-538
Methysergide
361-37-5
353.5
2 mg/ml
208
14.14554247
2749
4361
1.113161115


313
4-H4
DL-155
Fosinopril 
98048-97-6
563.7
2 mg/ml
267
8.870321611
3091
5290
1.11308584


203
3-E4
CA-237
Cilnidipine
132203-70-4
492.5
2 mg/ml
372 
10.1515956
2863
4352
1.112274868


618
8-F9
RA-104
Picotamide
32828-81-2
376.4
2 mg/ml
92
13.28308608
2800
4361
1.110016418


505
7-C6
DL-462
Omeprazole
73590-58-6
345.4
2 mg/ml
521
14.47499427
3182
4940
1.109359452


494
7-B5
DL-452
Myclobutanil
88671-89-0
288.8
2 mg/ml
468
17.31406615 
3430
4940
1.108435718


497 
7-B8
DL-455
Naphazoline HCl
835-31-4
210.3
2 mg/ml
491
23.77769793
3265
4940
1.108363804


64 
1-G5 
AC-238
Ivermectin
70288-86-7
875.1
2 mg/ml
48 
5.713524477
2997
5531
1.107715952


615
8-F6
PI-146
Argatroban
74863-84-6
508.6
2 mg/ml 
122
9.830046942
2317
4361
1.105957455


552
7-H3
DL-506
Streptomycin sulfate
3810-74-0
581.6
2 mg/ml
650 
8.597200325
3447
4940
1.104239856


467
6-G8
DL-425
Indapamide
26807-65-8
365.8
2 mg/ml
318
13.66713133
3873
4303
1.104220554


387
5-G8
DL-349
Chloramphenicol
56-75-7
323.1
2 mg/ml
331
15.47342379
3620
4930
1.103062547


155
2-H6
DL-119
Sodium phenylacetate
114-70-5
158.1
2 mg/ml
752 
31.61883569
3644
5627
1.102851143


604
8-E5
PD-140
Isobutylmethylxanthine
28822-58-4
222.2
2 mg/ml
271
22.49732147 
3315
4361
1.100601762


191
3-D2
C-115
Galanthamine HBr
1953-04-4
287.4
2 mg/ml
348 
17.39966708
3008
4352
1.100168157


131
2-F2
DL-248
Vardenafil
224785-90-4
488.6
2 mg/ml
721
10.23304135 
3824
5627
1.09750532


627
8-G8
DL-535
Efavirenz
154598-52-4
315.7
2 mg/ml
87 
15.83874736
2950
4361
1.096621218


610
8-E11
PD-185
Vinpocetine
42971-09-5
350.5
2 mg/ml
731
14.26677127
2743
4361
1.095654486


375
5-F6
DL-337
Betamethasone
378-44-9
504.6
2 mg/ml
177
9.908813352
3147
4930
1.095188192


480
6-H11
DL-438
Losartan potassium
124750-99-8
461.0
2 mg/ml
442
10.84562844
2989
4303
1.094220605


503
7-C4
DL-461
Ofloxacin
82419-36-1
361.4
2 mg/ml
515
13.83599695 
3273
4940
1.091241177


367
5-E8
DL-329
Apramycin 
37321-09-8
539.6
2 mg/ml
117
9.266332216
3565
4930
1.091102994


632 
8-H3
DL-107
Esmolol
81147-92-4
295.4 
2 mg/ml
217 
16.92723608
2801
4361
1.090858491


226
3-G7
EI-126
Dexamethasone
50-02-2
392.5
2 mg/ml
551
12.73976785
2885
4352
1.090773207


593
8-D4
NS-107
Dibenzepine HCl
315-80-0
295.4
2 mg/ml
342 
16.92691402
2911
4361
1.090214969


219
3-F10
D-111
Remoxipride
73220-03-8
371.3
2 mg/ml
615
13.4670228
3102
4352
1.089515817


231
3-H2
EI-160
Clofibrate
637-07-0
242.7
2 mg/ml
466
20.6011795
3202
4352
1.088970447


82
2-A3 
AC-615
Aminophylline
317-34-0
180.2
2 mg/ml
86 
27.75196691
3411
5627
1.086250792


577
8-B8
DL-158
Tropisetron HCl
105826-92-4
284.4
2 mg/ml
715
17.58327395
2870
4361
1.085917732


100
2-B11
AC-758
Risperidone
106266-06-2
410.5
2 mg/ml
622
12.18039734
3677
5627
1.085610636









Table 4-9 is a continuation of Table 4-8.


















TABLE 4-9







583
8-C4
DL-531
Iproniazid
54-92-2
179.2 
2 mg/ml
1
27.89813343 
2950
4361
1.083649788


112
2-D3
AC-812
Alprenolol HCl
13707-88-5
249.4
2 mg/ml
69
20.05164823
4059
5627
1.08286273


90
2-A11
AC-743
Neostigmine Br
114-80-7
223.3 
2 mg/ml
496
22.39165941
3700
5627
1.081134642


132
2-F3
DL-249
Linezolid
165800-03-3
337.4 
2 mg/ml
289
14.82123076
4094
5627
1.079830943


637
8-H8
DL-195
Trifluperidol 2HCl
749-13-3
409.4
2 mg/ml
709
12.21216805
2668
4361
1.076621099


361 
5-E2 
GR-341
Amifostine
20537-88-6
214.2
2 mg/ml
79
23.339979 
3793
4930
1.075525552


196
3-D7
CA-211
Nimodipine
66085-59-4
418.5 
2 mg/ml
142
11.94884261
3551
4352
1.074048725


633
8-H4
DL-123
Pantothenic acid
79-83-4
219.2
2 mg/ml
533
22.80610514
3095
4361
1.073870397


109 
2-C10
AC-809
Xylazine HCl
7361-61-7
220.3
2 mg/ml
734
22.69233478
4024
5627
1.073498572


139
2-F10
DL-255
Nisoldipine
63675-72-9
388.4
2 mg/ml
505
12.8725284
2492
5627
1.072692413


224 
3-G5 
EI-121
Disodium cromoglycate
15826-37-6
512.3 
2 mg/ml
746
9.759124699
3238
4352
1.072562348


184 
3-C5
NS-520
Melatonin
73-31-4
232.3 
2 mg/ml
187
21.52531202
3303
4352
1.068565791


182 
3-C3 
DL-279
Eriotinib
183321-74-6
393.4
2 mg/ml
196
12.70821424 
2238
4352
1.068496754


524
7-E5 
DL-481
Procarbazine HCl
366-70-1
221.3
2 mg/ml
560
22.59328122
3374
4940
1.067668516


360
5-D11
DL-565 
Sumatriptan Succinate
103628-48-4
413.5
2 mg/ml
76
12.092004
3805
4930
1.067073547


7 
1-A8 
A245
Lomofungin
26786-84-5
314.3 
2 mg/ml
349
15.91055721
2581
5531
1.066595684


50
1-E11
AC-181
Pindolol
13523-86-9
248.3
2 mg/ml
125
20.13468493
3750
5531
1.066333474


369
5-E10
AC-166 
Atenolol
29122-68-7
266.3
2 mg/ml
138
18.77278265
3690
4930
1.065359701


460
6-F11 
DL-418
Hexestrol
84-16-2
270.4
2 mg/ml
161
18.49284497
2460
4303
1.065264184


344
5-C5
DL-143
Thiamphenicol glycinate
2393-92-2
413.3
2 mg/ml
682
12.098354
3596
4930
1.063868092


578
8-B9
DL-526
Tylosin tartrate
1405-54-5
902.1
2 mg/ml
718
5.542647346
3044
4361
1.060390264


248
4-A9
EI-292
Meloxicam
71125-38-7
351.4
2 mg/ml
242
14.22858003
3513
5290
1.059662941


400
5-H11
AC-1290
Clopidogrel sulfate
135046-48-9
321.8
2 mg/ml
543
15.5362289
3664
4930
1.058515136


601
8-E2
NS-835 
Thioridazine HCl
130-61-0
370.6 
2 mg/ml
683
13.49226247
445
4361
1.057956033


483
7-A4 
DL-441
Mefenamic acid
61-68-7
241.3
2 mg/ml
149
20.72175532
3395
4940
1.057893663


608
8-E9
PD-179
Siguazodan
115344-47-3
284.3
2 mg/ml
628
17.58561963
3294
4361
1.054481673


424
6-C5 
DL-383
Enoxacin
74011-58-8
320.3
2 mg/ml
120
15.60909141
3281
4303 
1.053050564


403
6-A4 
DL-365
Corticosterone
50-22-6
346.5
2 mg/ml
43
14.43122849
3458
4303
1.051696556


371
5-F2
DL-333
Azaperone
1649-18-9
327.4 
2 mg/ml
150
15.27160131
3623
4930 
1.051506984









Table 4-10 is a continuation of Table 4-9.


















TABLE 4-10







98
2-B9
AC-755
Pergolide mesylate
66104-23-2
314.5
2 mg/ml 
415
15.89842964
3183
5627
1.051117975


83
2-A4
AC-626
Furafylline
80288-49-9
260.3
2 mg/ml 
292
19.2119681
3300
5627
1.047879978


51
1-F2
AC-183
Cimaterol
54239-37-1
219.3
2 mg/ml
374
22.80098312
4584
5531
1.047417124


307
4-G8
DL-102
Clindamycin palmitate
24729-96-2
425.0
2 mg/ml
454
11.76495198
3023
5290
1.045625788


214
3-F5
CT115
Shikonin
517-89-5
288.3
2 mg/ml 
610
17.34286978
2
4352
1.045253115


162
3-A3
DL-549
Meglumine
3521-84-4
195.2
2 mg/ml
182
25.61249201
3228
4352
1.044013498


421
6-C2
DL-181
Doxofylline
69975-86-6
266.3
2 mg/ml
33
18.77873405
3226
4303
1.044003177


564
8-A5
DL-206
Tibolone
5630-53-5
312.5
2 mg/ml
684
16.00224748
2584
4361
1.043057057


473
6-H4
DL-431
Levodopa
59-92-7
197.2
2 mg/ml 
251
25.35595707
3551
4303
1.042404777


437
6-D8
DL-395
Fenoldopam mesylate
67227-56-9
305.8
2 mg/ml 
81
16.35249062
3449
4303
1.041770576


154
2-H5
DL-209
Eprosartan
133040-01-4
424.5
2 mg/ml
160
11.77793236
3457
5627
1.0386449


567
8-A8
DL-517
Tobramycin (free base)
32986-56-4
467.5
2 mg/ml 
691
10.69464523
2799
4361
1.037850941


487
7-A8
DL-445
Metoprolol tartrate
56392-17-7
267.4
2 mg/ml
255
18.70057904
3197
4940
1.037553683


20
1-B11
AC-120
Glipizide
29094-61-9
445.5
2 mg/ml
437
11.22222511
3927
5531
1.036942137


526
7-E7
DL-483
Prednisone
53-03-2
358.4
2 mg/ml 
556
13.94939945
3551
4940
1.03678805


39
1-D10
AC-161
Yohimbine HCl
146-48-5
354.5
2 mg/ml
735
14.10624377
3881
5531
1.036293791


106
2-C7
AC-806
Idazoxan
79944-56-2
204.2
2 mg/ml
395
24.4821427
3950
5627
1.034852745


233
3-H4
EI-165
Benserazide HCl
322-35-0
257.2
2 mg/ml 
170
19.43648574
3528
4352
1.034730876


349
5-C10
T-117
Vincristine sulfate
2068-78-2
825.0
2 mg/ml
728
6.060752841
626
4930
1.034413759


135
2-F6
DL-258
Manidipine
120092-68-4
610.7
2 mg/ml 
472
8.187107366
3704
5627
1.034120443


117
2-D8
AC-840
Metoclopramide HCl
7232-21-5
299.8
2 mg/ml 
241
16.67759939
3640
5627
1.034082252


538
7-F9
DL-566
Nelfinavir Mesylate
159989-64-7
663.9
2 mg/ml 
623
7.531227709
3129
4940
1.033151485


628
8-G9
DL-536
Taurocholic acid, 
81-24-3
515.7
2 mg/ml
669
9.69527649
3078
4361
1.032215102





sodium salt hydrate










5
1-A6
A-239
Kasugamycin
6980-18-3
379.4
2 mg/ml 
68
13.17974363
3868
5531
1.031046544


63
1-G4
AC-232
Pancuronium Br
15500-66-0
572.9
2 mg/ml 
532
8.727839688
3597
5531
1.030984975


548
7-G9
DL-503
Sibutramine HCl
125494-59-9
279.9
2 mg/ml 
620
17.86630125
3356
4940
1.029548705


546
7-G7
DL-501
Scopolamine HBr
114-49-8
303.4
2 mg/ml
649
16.48200123
3410
4940
1.027058812


629
8-G10
SL-230
Miglustat
72599-27-0
219.3
2 mg/ml 
333
22.80156749
2966
4361
1.025463612


404
6-A5
DL-366
Crotamiton
483-63-6
203.3
2 mg/ml
488
24.5958242
3264
4303
1.025355545


65
1-G6
AC-241
Physostigmine sulfate
64-47-1
275.4
2 mg/ml
41
18.15847497
3672
5531
1.024400669









Table 4-11 is a continuation of Table 4-10.


















TABLE 4-11







312
4-H3
DL-108
Fluvastatin Na
93957-54-1
411.5
2 mg/ml
259
12.15133211
805
5290
1.024223587


70
1-G11
AC-779
Trans-triprolidine HCl
6138-79-0
278.4
2 mg/ml
704
17.95973205
4144
5531
1.02362497


122
2-E3
AC-911
Carbamylcholine Cl
51-83-2
147.2
2 mg/ml
335
33.96770283
3858
5627
1.023493355


180
3-B11
DL-277
Anagrelide
68475-42-3
256.1
2 mg/ml
103
19.52417325
4056
4352
1.023173844


138
2-F9
DL-254
Olmesartan
144689-63-4
558.6
2 mg/ml
519
8.950964023
3800
5627
1.022813346


280
4-D11
DL-236
Ergotamine D-tartrate
379-79-3
581.7
2 mg/ml
185
8.595829922
3583
5290
1.02240815


330
5-A11
DL-244
Lomerizine HCl
101477-55-8
468.5
2 mg/ml
346
10.67125274
1485
4930
1.022267701


432
6-D3
DL-170
Etretinate
54350-48-0
354.5
2 mg/ml
419
14.10462103
3065
4303
1.021125974


619
8-F10
S104
Conduritol b epoxide
6090-95-5
162.1
2 mg/ml
37
30.83686313
3331
4361
1.02104076


19
1-B10
AC-118
Pinacidil
85371-64-8
245.3
2 mg/ml
53
20.38072166
4040
5531
1.020537688


542
7-G3
DL-497
Roxithromycin
80214-83-1
837.1
2 mg/ml
639
5.97323558
3677
4940
1.020250018


394
5-H5
AR-116
Citalopram
59729-33-8
324.4
2 mg/ml
414
15.41299569
3008
4930
1.019988134


42
1-E3 
DL-564
Emtricitabine
143491-57-0
247.2
2 mg/ml
757
20.22247963
3766
5531
1.01940913


156
2-H7
DL-551
Ozagrel
82571-53-7
228.3
2 mg/ml
750
21.90553728
3862
5627
1.019096149


417
6-B8
DL-378
Diflunisal
22494-42-4
250.2
2 mg/ml
361
19.98371647
3527
4303
1.018838108


572
8-B3
DL-521
Tolmetin Na
64490-92-2
257.3
2 mg/ml
696
19.43319586
3245
4361
1.018708402


446
6-E7
DL-404
Fluocinolone acetonide
67-73-2
452.5
2 mg/ml
226
11.0497262
3099
4303
1.017955327


47
1-E8
AC-174
Clenbuterol
37148-27-9
277.2
2 mg/ml
90
18.03777442
4688
5531
1.017177264


356
5-D7
DL-318
Allopurinol
315-30-0
136.1
2 mg/ml
65
36.7339601
3361
4930
1.016923764


178
3-B9
PG-007
Dinoprostone
363-24-6
352.5
2 mg/ml
262
14.18540161
4302
4352
1.016831942


153
2-H4
DL-212
Escitalopram
128196-01-0
324.4
2 mg/ml
199
15.41299569
3381
5627
1.016098583


124
2-E5
AC-915
Butyrylcholine Cl
2963-78-2
174.3
2 mg/ml
277
28.69189354
4016
5627
1.01595962


145
2-G6
DL-261
Sildenafil
139755-83-2
474.6
2 mg/ml
634
10.53549387
3799
5627
1.014737397


260
4-B11
GR-210
Troglitazone
97322-87-7
441.6
2 mg/ml
712
11.32373333
3459
5290
1.011471642


322
5-A3
DL-544
Etoricoxib
202409-33-4
358.8
2 mg/ml
412
13.93341971
3601
4930
1.011198164


468
6-G9
DL-426
Itopride HCl
122892-31-3
358.4
2 mg/ml
28
13.94928853
3763
4303
1.010794493


426
6-C7
DL-385
Ethisterone
434-03-7
312.5
2 mg/ml
345
16.00224748
3339
4303
1.010472666


281
4-E2
DL-291
Sulindac
38194-50-2
356.4
2 mg/ml
662
14.02842929
3497
5290
1.010000074


410
6-A11
DL--372
Danazol
17230-88-5
337.5
2 mg/ml
188
14.81630786
3085
4303
1.00945183


173
3-B4
DL-273
Montelukast
158966-92-8
586.2
2 mg/ml
459
8.529525644
3436
4352
1.009136665









Table 4-12 is a continuation of Table 4-11.


















TABLE 4-12







386
5-G7
DL-348
Ceftazidime
72558-82-8
546.6
2 mg/ml
327
9.147714067
3569
4930
1.009027318


331
5-B2
DL-138
Lofexidine
21498-08-8
259.1
2 mg/ml
325
19.29480647
3658
4930
1.008563082


543
7-G4
DL-498
Rufloxacin
106017-08-7
363.4
2 mg/ml
641
13.75842425
3646
4940
1.00822304


550
7-G11
DL-504
Spironolactone
52-01-7
416.6
2 mg/ml
640
12.00237436
3332
4940
1.007768493


150
2-G11
DL-264
Candesartan
139481-59-7
440.5
2 mg/ml
298
11.35162944
3901
5627
1.006627197


40
1-D11
AC-164
Dihydroergotamine mesylate
11032-41-0
583.7
2 mg/ml
432
8.56614194
3623
5531
1.00594313


433
6-D4
DL-563
Trichloromethiazide
133-67-5
380.7
2 mg/ml
424
13.13516192
3381
4303
1.005709679


123
2-E4
AC-912
Carbamyl-beta-
590-63-6
161.2
2 mg/ml
332
31.01241774
3975
5627
1.005669411





methylcholine Cl










290
4-E11
J120
Mephenytoin
50-12-4
218.3
2 mg/ml
399
22.90871181
3501
5290
1.003983273


333
5-B4
DL-205
Nifekalant HCl
130636-43-0
405.5
2 mg/ml
503
12.33174749
3562
4930
1.003501259


250
4-A11
EI320
Sodium phenylbutyrate
1716-12-7
186.2
2 mg/ml
753
26.85461027
3595
5290
1.003310817


440
6-D11
DL-398
Finasteride
98319-26-7
372.6
2 mg/ml
109
13.42081666
3433
4303
1.002444372


431
6-D2
DL-390
Etidronate 2Na
7414-83-7
204.0
2 mg/ml
354
24.50817965
3366
4303
1.001714982


116
2-D7
DL-558
Amfebutamone
34911-55-2
239.7
2 mg/ml
742
20.85527395
3526
5627
1.001708034


200
3-D11
CA-225
Flunarizine-2HCl
30484-77-6
404.5
2 mg/ml
211
12.36071575
3062
4352
1.000484085


326
5-A7
DL-213
Ibandronate
114084-78-5
319.2
2 mg/ml
230
15.66250387
3747
4930
0.999589951


351
5-D2
DL-313
Acetylsalicylic acid
50-78-2
180.2
2 mg/ml
57
27.75284493
3375
4930
0.997680094


393
5-H4
DL-355 
Ciprofloxacin
85721-33-1
331.3
2 mg/ml
386
15.08979742
3892
4930
0.997536588


1
1-A2
DL-552
Moroxydine HCl
3160-91-6
207.7
2 mg/ml
748
24.07731167
3878
5531
0.99722472


563
8-A4
DL-190
Temozolomide
85622-93-1
194.2
2 mg/ml
674
25.75279011
2729
4361
0.996678344


372
5-F3
T-116
Vinblastine sulfate
143-67-9
811.0
2 mg/ml
727
6.165254264
741
4930
0.994519761


161
3-A2
AR-103
Tizanidine HCl
51322-75-9
253.7
2 mg/ml
690
19.70718202
3281
4352
0.99376949


58
1-F9
AC-214
Pilocarpine HCl
54-71-7
208.3
2 mg/ml
102
24.00817776
3649
5531
0.992948705


227
3-G8
EI-127
Dipyridamole
58-32-2
504.6
2 mg/ml
314
9.908100387
4027
4352
0.992730979


237
3-H8
EI-180
Neomycin sulfate
1405-10-3
614.7
2 mg/ml
494
8.134638418
3484
4352
0.992072921


215
3-F6
D-102
Bromocriptine mesylate
22260-51-1
654.6
2 mg/ml
238
7.638134517
3228
4352
0.991102429


125
2-E6
AC-925
Famotidine
76824-35-6
337.4
2 mg/ml
456
14.81716581
3670
5627
0.990592254


43
1-E4
AC-169
Betaxotol HCl
63659-19-8
307.4
2 mg/ml
179
16.26350892
3991
5531
0.990435828


501
7-C2
DL-459
Norfloxacin
70458-96-7
319.3
2 mg/ml
178
15.65736377
3625
4940
0.990112519









Table 4-13 is a continuation of Table 4-12.


















TABLE 4-13







190
3-C11
C-112
Gallamine triethiodide
65-29-2
510.8
2 mg/ml
352
9.78797293
3481 
4352
0.989145605


561
8-A2 
DL-514
Tetracycline
60-54-8
444.4 
2 mg/ml
680
11.24998138
3102
4361
0.988225706


384
5-G5 
DL-346 
Cefoperazone acid 
62893-19-0
645.7
2 mg/ml 
340
7.743804708
4017
4930
0.987788853


96
2-B7 
AC-751
Domperidone
57808-66-9
425.9 
2 mg/ml
239
11.73924195
3927
5627
0.987539696


354
5-D5
DL-316
Acycloguanosine
59277-89-3
225.2
2 mg/ml
59
22.20164179
3386
4930
0.987434293


208
3-E9 
DL-281
Latanoprost
130209-82-4
432.6
2 mg/ml
168
11.55786898
3355
4352
0.987252914


327
5-A8
DL-202
Imipenem
64221-86-9
299.4
2 mg/ml
413
16.70280095 
3759 
4930 
0.98696121


89
2-A10
AC-741
Mecamylamine HCl
826-39-1
167.3 
2 mg/ml
143
29.88701751
3575
5627
0.986404772


423
6-C4
PI-152
Enalapril
75847-73-3
376.5
2 mg/ml
114
13.28174491
3515
4303
0.98624125


212
3-F3
CR110
Pravadoline
92623-83-1
378.5
2 mg/ml
410
13.21090279
3403
4352
0.985697957


30 
1-C11
AC-136
Cirazoline HCl
40600-13-3
216.3
2 mg/ml
389
23.11761684 
3800 
5531
0.984871977


496
7-B7
DL-454
Nabumetone
42924-53-8
228.3
2 mg/ml
22
21.9016243
3581 
4940 
0.98484544


192
3-D3
CA-200
Amiloride
2016-88-8
229.6 
2 mg/ml 
85
21.77416222
3198 
4352
0.983589974 


240
3-H11
EI-216
Docebenone
80809-81-0
326.4
2 mg/ml
453
15.31677057
2365
4352
0.98213773


157 
2-H8
DL-217
Entacapone
130929-57-6
305.3
2 mg/ml
156
16.37772269
3918
5627
0.981956978


336
5-B7
DL-196
Pamidronic acid
40391-99-9
235.1
2 mg/ml
531
21.27014953
3882
4930
0.981721922


311
4-H2
DL-192
Enalaprilat
76420-72-9
348.4 
2 mg/ml
115
14.35122228
3680 
5290
0.981394891


53 
1-F4
AC-189
Pronethalol HCl
51-02-5
229.3
2 mg/ml
564
21.80313876
3891
5531
0.981058038


249
4-A10 
EI-318
Terbinafine HCl
91161-71-6
291.4 
2 mg/ml
759
17.15618407
2570
5290
0.979599083


293 
4-F4
KC-125
Minoxidil
38304-91-5
209.3
2 mg/ml
404
23.89454287
3057
5290
0.979418493


454
6-F5
DL-412
Gatifloxacin
112811-59-3
375.4
2 mg/ml 
376
13.31900954
3536
4303
0.979414164


80
1-H11
AC-508
Propofol
2078-54-8
178.3
2 mg/ml 
593
28.04629613
3615 
5531
0.979366862


189
3-C10
C-110
Tacrine HCl 
1684-40-8
198.3 
2 mg/ml
665
25.21814579
3237 
4352
0.979114411


581
8-C2
DL-529
Venlafaxine HCl
99300-78-4
277.4 
2 mg/ml
724
18.023848
2866 
4361
0.97800413


453
6-F4
DL-411
Ganciclovir
82410-32-0
255.2
2 mg/ml
585
19.5897852
3183 
4303
0.977523039


220 
3-F11
DM100
Calcifediol
19356-17-3
400.7
2 mg/ml
280
12.47970395
744
4352 
0.977023901


479
6-H10
DL-437
Lorglumide
97964-56-2
459.4
2 mg/ml
430
10.88335064 
3404
4303
0.976756084


71
1-H2
AC-280
Cimetidine
51481-61-9
252.3
2 mg/ml
379
19.8142831
3951
5531
0.976328942


239
3-H10
EI-213
Captopril
62571-86-2
217.3
2 mg/ml
311
23.01085008
120
4352
0.976258428


442
6-E3 
DL-400
Flurbiprofen
5104-49-4
244.3
2 mg/ml
234
20.46931555
3303
4303
0.975748109









Table 4-14 is a continuation of Table 4-13.


















TABLE 4-14







472
6-H3
DL-430
Levonorgestrel
797-63-7
312.5 
2 mg/ml
589
16.00224748
4342
4303
0.973379693


438
6-D9
DL-396
Fenoprofen
31879-05-7
242.3 
2 mg/ml
88
20.63753217
3807
4303
0.973324462


247
4-A8
EI-288
Keloprofen
22071-15-4
254.3
2 mg/ml
83
19.66273069
3489
5290
0.971845965


456
6-F7
DL-414
Gemfibrozil
25812-30-0
250.3
2 mg/ml
394
19.97277391
3485
4303
0.971302374


397
5-H8
DL-359
Clinafloxacin HCl
105956-99-8
365.8 
2 mg/ml
444
13.66886796
3716
4930
0.970430289


33
1-D4
AC-144
Naftopidil 2HCl
57149-07-2
392.5
2 mg/ml
485
12.73877691
3686
5531
0.968951371


81
2-A2
AC-554
Mianserin hcl
21535-47-7
264.4
2 mg/ml 
305
18.91263686
3669
5627
0.968915746


22
1-C3
AC-122
Quinine
6119-47-7
324.4
2 mg/ml
595
15.41181225
4145
5531
0.968822029


87
2-A8
AC-735
Atropine sulfate
55-48-1
289.4
2 mg/ml
506
17.27845291
3837
5627
0.96866694


91
2-B2
AC-745
Strychnine HCl
1421-86-9
334.4
2 mg/ml
652
14.95118334
3678
5627
0.968275526


597
8-D8
NS-145
Ondansetron
99614-01-4
293.4
2 mg/ml 
263
17.04322944
3376
4361
0.968267341


68
1-G9
AC-259
Piribedil HCl
78213-63-5
298.3
2 mg/ml
312
16.75898297
3965
5531
0.968074637


256
4-B7
G-430
Suramin sodium
129-46-4
1291.3
2 mg/ml
664
3.872216754
3560
5290
0.967958181


414
6-B5
DL-375
Diclofenac, Na
15307-79-6
296.2
2 mg/ml
357
16.88303571
3436
4303
0.967481903


228
3-G9
EI-128
Ethacrynic acid
58-54-8
303.1
2 mg/ml
321
16.49380118
3818
4352
0.966834893


385
5-G6
DL-347
Cefotamine acid
63527-52-6
455.5
2 mg/ml
326
10.97764274
3475
4930
0.966732389


104
2-C5
AC-766
Ranitidine HCl
66357-59-3
314.4
2 mg/ml
613
15.90283799
3674
5627
0.966680475


366
5-E7
DL-328
Ampiroxicam
99464-64-9
447.5
2 mg/ml
99
11.17392671
3560
4930
0.966190326


606
8-E7
PD-152
Milrinone
78415-72-2
211.2
2 mg/ml
355
23.67143705
3200
4361
0.965482734


396 
5-H7 
DL-358
Climbazole
38083-17-9
292.8
2 mg/ml
440
17.07837272
3594
4930
0.964692099


499
7-B10
DL-457
Niflumic acid
4394-00-7
282.2
2 mg/ml
504
17.71641881
3494
4940
0.963223749


252
4-B3
FR-112
Ambroxol
18683-91-5
378.1
2 mg/ml
77
13.2236927
3207
5290
0.96203255


232
3-H3
EI-164
Ibuprofen
15687-27-1
206.3
2 mg/ml
435
24.2380514
3501
4352
0.961279855


338
5-B9
DL-310
Triptorelin
57773-63-4
1311.5
2 mg/ml
711
3.812486831
3781
4930
0.960932891


102
2-C3
AC-763
Diphenhydramine HCl
147-24-0
255.4
2 mg/ml 
282
19.57996894
3990
5627
0.960654141


320
4-H11
DL-546
Atazanavir
198904-31-3
704.9
2 mg/ml
743
7.093464941
3546
5290
0.960442125


283
4-E4
DL-547
Calcipotriene
112965-21-6
412.6
2 mg/ml
281
12.11774424
1637
5290
0.95970899


18
1-B9
AC-117
Tolbutamide
64-77-7
270.4
2 mg/ml
693
18.49434846
3936
5531
0.959672646


341
5-C2
DL-187
Rocuronium bromide
119302-91-9
529.8
2 mg/ml
630
9.43769799
3753
4930
0.957592971


28
1-C9
AC-130
Flecainide
54143-56-5
414.4
2 mg/ml
112
12.06706537
4348
5531
0.957511107









Table 4-15 is a continuation of Table 4-14.


















TABLE 4-15







201
3-E2
CA-234
Gabapentin
60142-96-3
171.2
2 mg/ml
341
29.19855684
3385
4352
0.956610942


38
1-D9
AC-160
Spiroxatrine
1054-88-2
379.5
2 mg/ml
644 
13.17652044
3716
5531
0.954990916


350
5-C11
DL-312
Acemetacin
53164-05-9
415.8
2 mg/ml
49 
12.0240372
3440
4930
0.953477871


346
5-C7
NP-461
Vinorelbine
71486-22-1
779.0
2 mg/ml 
730
6.418863201
756
4930
0.952859497


198
3-D9
CA-215
Verapamil
23313-68-0
454.6
2 mg/ml 
725
10.99831943
2810
4352
0.951456917


66
1-G7
DL-554
Nialamide
51-12-7
298.3
2 mg/ml
749
16.75898297
3940
5531
0.951275207


78
1-H9
AC-439
Aniracetam
72432-10-1
219.2
2 mg/ml
113
22.805817
4429
5531
0.950109296


488
7-A9
DL-446
Methimazole
60-56-0
114.2
2 mg/ml 
64
43.79441082
3779
4940
0.95010631


434
6-D5
DL-392
Famciclovir
104227-87-4
321.3
2 mg/ml
450
15.55990949
3588
4303
0.94998846


316
4-H7
DL-166
Granisetron
109889-09-0
312.4
2 mg/ml
474
16.00419028
3431
5290
0.94970961


308
4-G9
DL-164
Diclazuril
101831-37-2
407.6
2 mg/ml
343
12.26554773
3898
5290
0.949157619


74
1-H5
DL-556
Zoledronic acid
118072-93-8
272.1
2 mg/ml
755
18.3761206
3455
5531
0.948434746


23
1-C4
AC-123
Flufenamic acid 
530-78-9
281.2
2 mg/ml
204
17.77863115
3954
5531
0.947507357


519
7-D10
DL-476
Piperacillin
61477-96-1
517.6
2 mg/ml
571
9.660623457
3579
4940
0.947504161


474
6-H5
DL-432
Levofloxacin HCl
100986-85-4
361.4
2 mg/ml
274
13.83599695
3231
4303
0.947418002


512
7-D3
NS-710
Paroxetine HCl
110429-49-8
329.4
2 mg/ml
534
15.18028639
3364
4940
0.947243065


136
2-F7
DL-252
Tolcapone
134308-13-7
273.2
2 mg/ml
694
18.29843109
3782
5627
0.946861128


557
7-H8
DL-160
Tamsulosin HCl
106463-17-6
408.5
2 mg/ml
667
12.23928607
3550
4940
0.946457969


448
6-E9
DL-406
Flutamide
13311-84-7
276.2
2 mg/ml 
237 
18.10170202
3363
4303
0.944941502


340
5-B11
DL-198
Risedronic acid
105462-24-6
283.1
2 mg/ml
618
17.66061678
3396
4930
0.944480182


27
1-C8
AC-128
Lidocaine
6108-05-0
234.3
2 mg/ml
284
21.33613354
3924
5531
0.944395213


37
1-D8
AC-154
Rilmenidine
54187-04-1
180.3
2 mg/ml
629
27.73897096
3957
5531
0.943079626





hemifumarate










301
4-G2
DL-301
Abamectin
71751-41-2
873.1
2 mg/ml 
3
5.72672088
3309
5290
0.942934361


67
1-G8
AC-250
Haloperidol HCl
52-86-8
375.9
2 mg/ml 
78 
13.3023173
3283
5531
0.94230727


209 
3-E10
CM-109
Ouabain
11018-89-6
584.7
2 mg/ml
279
8.551879025 
3390
4352
0.942018317


343
5-C4
DL-176
Sulbactam
68373-14-8
233.2
2 mg/ml
656
21.43672627
3575
4930
0.941264997


566
8-A7
DL-516
Tinidazole
19387-91-8
247.3
2 mg/ml
687
20.22044245
3225
4361
0.940938995


59
1-F10
AC-218
Ipratropium Br
22254-24-6
332.5
2 mg/ml 
225
15.03908659
4011
5531
0.940156802


462
6-G3
DL-420
Hydrocortisone 21-
50-03-3
404.5
2 mg/ml 
192
12.36069833
3649
4303
0.939505109





acetate

















Table 4-16 is a continuation of Table 4-15.


















TABLE 4-16







428
6-C9
BL-093
Estradiol
50-28-2
272.4
2 mg/ml
244
18.35598107
2656
4303
0.938889093


126
2-E7
AC-928
Terfenadine
50679-08-8
471.7
2 mg/ml
678
10.60020322
279
5627
0.938608243


25
1-C6
AC-125
Phenytoin
57-41-0
252.3
2 mg/ml
546
19.81963419
3963
5531
0.936703079


129
2-E10
DL-246
Amorolfine
78613-35-1
317.5
2 mg/ml
98
15.74707923
3844
5627
0.933498688


580
8-B11
DL-528
Vecuronium Br
50700-72-6
557.8
2 mg/ml
723
8.963073018
3273
4361
0.931794456


335
5-B6
DL-194
Oseltamivir
196618-13-0
312.4
2 mg/ml
522
16.00447818
3897
4930
0.930834833


388
5-G9
DL-350
Chlormadinone acetate
302-22-7
449.0
2 mg/ml
362
11.13606183
4112
4930
0.930559729


401
6-A2
DL-363
Clobetasol propionate 
25122-46-7
467.0
2 mg/ml
460
10.70704645
3051
4303
0.930434551


362
5-E3
DL-324
DI-aminoglutethimide
125-84-8
232.3
2 mg/ml
418
21.52531202
3682
4930
0.929172021


545
7-G6
DL-500
Scopolamine n-butylbromide
149-64-4
360.5
2 mg/ml
651
13.8704893
3414
4940
0.928722757


379
5-F10
DL-341
Buspirone HCl
33386-08-2
385.5
2 mg/ml
252
12.96971375
3914
4930
0.925652197


363
5-E4
DL-325
4-aminosalicylic acid
65-49-6
153.1
2 mg/ml
31
32.65013151
3927
4930
0.925638387


622
8-G3
S-520
Melengestrol acetate
2919-66-6
396.5
2 mg/ml
223
12.60934223
3402
4361
0.924253635


257
4-B8
DL-282
Goserelin acetate
145781-92-6
1269.4
2 mg/ml
447
3.938746102
3481
5290
0.924156734


137
2-F8
DL-253
Gestrinone
16320-04-0
308.4
2 mg/ml
406
16.21143708
3951
5627
0.92396976


167
3-A8
B-100
Flumazenil
78755-81-4
333.3
2 mg/ml
205
16.48557102
3334
4352
0.92218228


9
1-A10
A-256
Novobiocin Na
1476-53-5
612.6
2 mg/ml
513
8.161408236
3901
5531
0.921040001


353
5-D4
DL-315
Aceclofenac
89796-99-6
354.2
2 mg/ml
45
14.11662495
3872
4930
0.920926571


229
3-G10
EI-131
Indomethacin
53-86-1
357.8
2 mg/ml
480
13.97441737
3625
4352
0.920711275


77
1-H8
AC-408
Memantine HCl
19982-08-2
179.3
2 mg/ml
383
27.88500025
3630
5531
0.915736222


143
2-G4
NA-137
Lamotrigine
84057-84-1
256.1
2 mg/ml
131
19.52395597
3616
5627
0.915452963


79
1-H10
AC-501
Riluzole HCl
1744-22-5
234.2
2 mg/ml
631
21.34917841
3088
5531
0.914509341


204
3-E5
CA-305
Phenoxybenzamine HCl
63-92-3
303.8
2 mg/ml
417
16.45629271
3736
4352
0.912899056


504
7-C5
DL-106
Oltipraz
64224-21-1
226.3
2 mg/ml
520
22.09042388
3600
4940
0.909397243


449
6-E10
DL-407
Fluconazole 
86386-73-4
306.3
2 mg/ml
203
16.32509187
3591
4303
0.908986259


213
3-F4
CT110
Tranilast
53902-12-8
327.3
2 mg/ml
702
15.27464312
3720
4352
0.907366883


315
4-H6
DL-243
Ginkgolide a
15291-75-5
408.4
2 mg/ml
416
12.24263292
3561
5290
0.906948111


242
4-A3
EI-219
Piroxicam
36322-90-4
331.4
2 mg/ml
169
15.08966763
3793
5290
0.905024596


297
4-F8
DL-221
Bromebric acid
16170-76-6
285.1
2 mg/ml
231
17.53795336
3911
5290
0.904246558









Table 4-17 is a continuation of Table 4-16.


















TABLE 4-17







183
3-C4
AC-227
Lobeline
90-69-7
337.5
2 mg/ml
315 
14.81630786
3100
4352
0.903196994


223
3-G4
EI-114
Oxatomide
60607-34-3
426.6
2 mg/ml
525
11.72150593
3155
4352
0.90238694


402
6-A3
DL-364
Closantel
57808-65-8
663.1
2 mg/ml
17
7.540524134
3506
4303
0.900918749


277
4-D8
DL-287
Pranlukast
103177-37-3
481.5
2 mg/ml 
209
10.38388259
3435
5290
0.900459646


288
4-E9
DL-161
Bambuterol
81732-65-2
367.4
2 mg/ml
166
13.60733313
3715
5290
0.898480586


595
8-D6
NS-109
Fluperlapine
67121-76-0
309.4
2 mg/ml
229
16.16084566
3074
4361
0.898284379


605
8-E6
PD-141
Irsogladine maleate
57381-26-7
256.1
2 mg/ml
670
19.52395597
3312
4361
0.898268861


415
6-B6
DL-376
2′,3′-dideoxycytidine
7481-89-2
211.2
2 mg/ml
6
23.67174748
3475
4303
0.89779101


218
3-F9
D-109
Quinpirole HCl (−)-
85760-74-3
219.3
2 mg/ml
599
22.79644751
3761
4352
0.896944187


452
6-F3
DL-410
Furosemide
54-31-9
330.7
2 mg/ml
294
15.11720962
3895
4303
0.895842738


84
2-A5
DL-561
nateglinide
105816-04-4
317.4
2 mg/ml
566
15.751408
3809
5627
0.894848357


35
1-D6
AC-152
Clonidine HCl
4205-91-8
230.1
2 mg/ml
469
21.72985462
4136
5531
0.894201589


253
4-B4
FR114
Idebenone
58186-27-9
338.4
2 mg/ml
431
14.77332039
2486
5290
0.893135387


420
6-B11
DL-380
Doxifluridine
3094-09-5
246.2
2 mg/ml
446
20.30895444
3672
4303
0.888312524


599
8-D10
DL-562
Tiotropium Br
136310-93-5
472.4
2 mg/ml
167
10.58372436
3450
4361
0.885587182


406
6-A7
DL-368
Cyproterone acetate
427-51-0
416.9
2 mg/ml
587
11.99186481
3604
4303
0.883618335


395
5-H6
DL-357
Clarithromycin
81103-11-9
748.0
2 mg/ml
426
6.684737473
3782
4930
0.881779446


207
3-E8
DL-131
Miltefosine
58066-85-6
407.6
2 mg/ml
369
12.26756431
3677
4352
0.880978036


358
5-D9
DL-320
Altretamine
645-05-6
210.3
2 mg/ml
548
23.77736662
4029
4930
0.880446665


427
6-C8
DL-386
Esomeprazole
119141-88-7
345.4
2 mg/ml
240
14.47499427
3677
4303
0.879982815


305
4-G6
DL-162
Bicalutamide
90357-06-5
430.4
2 mg/ml
186
11.61761647
3892
5290
0.879476573


2
1-A3
A-190
Clindamycin HCl
21462-39-5
425.0
2 mg/ml
445
11.76495198
3858
5531
0.878795179


430
6-C11
BL-090
Estrone
53-16-7
270.4
2 mg/ml
300
18.49284497
3156
4303
0.878561752


31
1-D2
AC-138
Oxymetazoline HCl
2315-02-8
260.4
2 mg/ml
350
19.20252085
4199
5531
0.878314397


15
1-B6
AC-109
Pimozide
2062-78-4
461.6
2 mg/ml
425
10.83283649
3602
5531
0.877950741


508
7-C9
DL-465
Oxacillin sodium
7240-38-2
400.4
2 mg/ml
293
12.48637705
3767
4940
0.877909406





monohydrate










364
5-E5
DL-326
5-aminosalicylic acid
89-57-6
153.1
2 mg/ml
34
32.65013151
3928
4930
0.877441514


188
3-C9
C-109
Oxotremorine 
17360-35-9
206.3
2 mg/ml
344
24.23771654
3867
4352
0.87722537





sesquifumarate










151
2-H2
DL-101
Butenafine
101828-21-1
317.5
2 mg/ml
276
15.74910591
3592
5627
0.87655312









Table 4-18 is a continuation of Table 4-17.


















TABLE 4-18







285
4-E6
DL-295
Zileuton
111406-87-2
236.3
2 mg/ml 
739
21.16003636
3595
5290
0.875950257


291
4-F2
KC-115
Diazoxide
364-98-7
230.7
2 mg/ml
322
21.67559362
3792
5290
0.875618181


12
1-B3
AC-104
Bepridil
74764-40-2
366.6
2 mg/ml
511
13.64065251
2664 
5531
0.874980688


339
5-B10
NP-526
Rifamycin sv
6998-60-3
696.8
2 mg/ml
584
7.175879824
3821
4930
0.873727406


300
4-F11
DL-300
Letrozole
112809-51-5
285.3
2 mg/ml 
180
17.52475336
3725
5290
0.873316695


282
4-E3
DL-292
Valproic acid
99-66-1
144.2
2 mg/ml
720
34.67033229
3654
5290
0.872956284


193 
3-D4
CA-202
Amlodipine
88150-42-9
408.9
2 mg/ml
97
12.22835773
3638
4352
0.872263773


317
4-H8
DL-105
Nedaplatin
95734-82-0
303.2
2 mg/ml
493
16.49143636
3606
5290
0.871858344


44
1-E5
AC-171
Practolol
6673-35-4
266.3
2 mg/ml 
126
18.77278265
4078
5531
0.867690682


458
6-F9
DL-416
Glimepiride
93479-97-1
490.6
2 mg/ml
436
10.19105414
3728
4303 
0.866489362


463
6-G4
DL-421
17-hydroxyprogesterone
68-96-2
330.5
2 mg/ml 
14
15.12990003
3290
4303
0.865994297


211
3-F2
CN-244
Molsidomine
25717-80-0
242.2
2 mg/ml
455
20.64099964
3514
4352
0.86575851


592
8-D3
NS-102
Cinanserin
1166-34-3
340.5
2 mg/ml 
384
14.6846725
3293
4361
0.865527858


292
4-F3
KC-120
Glyburide
10238-21-8
494.0
2 mg/ml
441
10.1211766
3706
5290
0.865161801


46
1-E7
AC-173
Xamoterol hemifumarate
73210-73-8
339.4
2 mg/ml
733
14.73210767
4274
5531
0.864243199


29
1-C10
AC-131
Phenamil
2038-35-9
305.7
2 mg/ml
545
16.35436695
4494
5531
0.864179707


181
3-C2
DL-278
Dofetilide
115256-11-6
441.6
2 mg/ml 
146
11.32317813
3730
4352
0.863856218


455
6-F6
DL-413
Gentamycin sulfate
1405-41-0
477.6
2 mg/ml
398
10.4688767
3459
4303
0.86296421


101
2-C2
AC-760
Sulpiride s (−)
23672-07-3
341.4
2 mg/ml
663
14.64419326
3782
5627 
0.862856671


374
5-F5
DL-336
Aztreonam
78110-38-0
435.4
2 mg/ml 
164
11.48271367
3727
4930 
0.862363652


186
3-C7
EI-233
Fasudil
103745-39-7
291.4
2 mg/ml
457
17.1600472
3468
4352 
0.860413294


3
1-A4
A-192
Clindamycin PO4
35208-55-0
663.4
2 mg/ml 
448
7.536839961
4024
5531
0.860199544


590
8-C11
NO-102
Naloxonazine 2HCl
82824-01-9
650.8
2 mg/ml 
38
7.683068625
3142
4361
0.859376357


14
1-B5
AC-108
Nicardipine
54527-84-3
479.5
2 mg/ml
497
10.42671259
4070
5531
0.857242752


491
7-B2
DL-449
Minocycline HCl 
10118-90-8
457.5
2 mg/ml
390
10.92925941
3723
4940
0.855177004


179
3-B10
DL-276
Metformin
657-24-9
129.2
2 mg/ml
2
38.70994614
3788
4352
0.8550975


352
5-D3
DL-314
Acipimox
51037-30-0
154.1
2 mg/ml
58
32.44092248
3854
4930
0.854913554


465
6-G6
DL-423
Ifosfamide
3778-73-2
261.1
2 mg/ml 
347
19.15051385
3845
4303
0.853784324


6
1-A7
A-240
Lincomycin
154-21-2
406.5
2 mg/ml
290
12.29874699
3884
5531
0.85175097









Table 4-19 is a continuation of Table 4-18.


















TABLE 4-19







516
7-D7
DL-473
Pentoxifylline 
6493-05-6
278.3 
2 mg/ml
538
17.96535783
3878
4940
0.850803206


41
1-E2
AC-165
Nicergoline
27848-84-6
484.4
2 mg/ml
575
10.32210926
3812
5531
0.850742508


21
1-C2
AC-121
PhentolamineHCl
73-05-2
281.4 
2 mg/ml
420
17.77079709
3946
5531
0.848862717


11
1-B2
A-280
Spectinomycin
21736-83-4
332.4
2 mg/ml
632
15.04408307
3970
5531
0.848019914


88
2-A9
AC-737
Decamethonium 2Br
541-22-0
258.5
2 mg/ml
550
19.34275934
3676
5627
0.848001798


72
1-H3
AC-282
Tiotidine
69014-14-8
312.4
2 mg/ml
689
16.00406529
3955
5531
0.847439599


391
5-H2
DL-353
Chloroquine phosphate
50-63-5
319.9
2 mg/ml
363
15.63081173
1425
4930
0.847341595


477
6-H8
DL-435
Lomefloxacin HCl
98079-52-8
351.4
2 mg/ml
154
14.23057771
3854
4303
0.845872371


295
4-F6
KC-152
Nicorandil
65141-46-0
211.2
2 mg/ml
574
23.67663811
3867
5290
0.845476715


210
3-E11
CM-112
Clopamide
636-54-4
345.9
2 mg/ml
471
14.45710115
3791
4352
0.845450133


194
3-D5
CA-205
Diltiazem
33286-22-5
414.5
2 mg/ml
477
12.06192535
3713
4352
0.844922239


310
4-G11
DL-199
Dolasetron
115956-12-2
324.4
2 mg/ml
235
15.41388517
3630
5290
0.844028477


306
4-G7
DL-180
Clodronate disodium
10596-23-3
244.9
2 mg/ml
465
20.41707761
3699
5290
0.842725479


265
4-C6
GR-306
Rifampicin
38776-75-9
823.0
2 mg/ml
760
6.075619764
3879
5290
0.841018412


230
3-G11
EI-133
Naproxen
22204-53-1
230.3
2 mg/ml
492
21.71402902
3884
4352
0.84034922


148
2-G9
DL-191
Celepime
88040-23-7
480.6
2 mg/ml
339
10.4043477
4053
5627
0.83920257


383
5-G4
DL-345
Carbamazepine
298-46-4
236.3
2 mg/ml
324
21.16172002
3941
4930
0.837664289


598
8-D9
NS-515
Ketanserin tartrate
74050-98-9
395.4 
2 mg/ml
71
12.64422829
3251
4361
0.837059608


175
3-B6
DL-275
Vatalanib 
212141-54-3
346.8
2 mg/ml
722
14.41660262
2326
4352
0.835361434


187
3-C8
C-106
Hydroxytacrine maleate
112964-99-5
214.3
2 mg/ml
227
23.33511754
4056
4352
0.834268975


584
8-C5
NA-103
Benzamil
2898-76-2
319.8
2 mg/ml
510
15.63693147
2764
4361
0.833088485


141
2-G2
DL-257
Olanzapine
132539-06-1
312.4
2 mg/ml
516
16.00308539
3911
5627
0.832761409


174
3-B5
EI-240
Selegiline
14611-51-9
187.3
2 mg/ml
655
26.6969745
3674
4352
0.831374618


284
4-E5
DL-294
Zafirlukast
107753-78-6
575.7
2 mg/ml
736
8.685242571
3543
5290
0.831323412


621
8-G2
S-515
Megestrol acetate 
595-33-5
384.5
2 mg/ml
580
13.00321668
3787
4361
0.830933611


45
1-E6
AC-172
Timolol maleate (s)
26921-17-5
316.4
2 mg/ml
686
15.80152126
4063
5531
0.82909403


576
8-B7
DL-525
Trimethoprim 
738-70-5
290.3
2 mg/ml
710
17.22210481
3404
4361
0.828969825


121
2-E2
AC-893
Nalbuphine HCl
59052-16-3
357.5
2 mg/ml
427
13.9878279
4002
5627
0.828433745


32
1-D3
AC-142
Ifenprodil
23210-56-2
325.5
2 mg/ml
273
15.36311459
3982
5531
0.827639882


319
4-H10
DL-174
Anetnole, trans-
104-46-1
148.2
2 mg/ml
703
33.73670285
3762
5290
0.827063694









Table 4-20 is a continuation of Table 4-19.


















TABLE 4-20







113
2-D4
AC-613
Imipramine HCl
113-52-0
280.4 
2 mg/ml
428
17.8306179
3986
5627
0.826869492


105
2-C6
AC-770
Naltrindole HCl
111469-81-9
414.5 
2 mg/ml
490
12.06247241
3917
5627
0.826560457


527
7-E8
DL-484
Pregnenolone
145-13-1
316.5 
2 mg/ml
557
15.7983878
3448
4940
0.825083643


26
1-C7
AC-127
Procainamide
614-39-1
235.3 
2 mg/ml
559
21.24659555
4211
5531
0.821713595


585
8-C6
NA-139
Bupivacaine HCl
14252-80-3
288.4
2 mg/ml
30
17.33482838
3039
4361
0.820582612


269
4-C10
GR-314
Delavirdine mesylate
147221-93-0
456.6
2 mg/ml
269
10.95120262
3382
5290
0.818777419


4
1-A5
A-195
Cyclosporin a
59865-13-3
1202.6
2 mg/ml
70
4.157511584
3693
5531
0.816836509


57
1-F8
DL-553
Canthaxanthin
514-78-3
564.9
2 mg/ml 
745
8.851762786
3915
5531
0.816437529


587
8-CB
NH-106
Ketotifen fumarate
34580-14-8
309.4
2 mg/ml
116
16.15856699
3463
4361
0.815032987


275
4-D6
DL-285
Methyl salicylate
119-36-8
152.2 
2 mg/ml
73
32.86205639
3675
5290
0.814008317


409
6-A10
DL-371
Dacarbazine
4342-03-4
182.2
2 mg/ml
184
27.44444969
3830
4303
0.813386494


489
7-A10
DL-447
Metronidazole
443-48-1
171.2
2 mg/ml
577
29.21295679
3748
4940
0.812670053


574
8-B5
DL-523
Tramadol HCl
27203-92-5
263.4 
2 mg/ml
701
18.98375048
3747
4361
0.81261827


149
2-G10
DL-178
Aripiprazole
129722-12-9
448.4 
2 mg/ml
133
11.1508443
3240
5627
0.818876527


429
6-C10
DL-388
Estriol
50-27-1
288.4
2 mg/ml
245
17.33762224
3711
4303
0.810492454


348
5-C9
DL-210
Salmeterol
89365-50-4
415.6
2 mg/ml 
645
12.03143747
4325
4930
0.810094503


309
4-G10
DL-302
Didanosine
69655-05-6
236.2
2 mg/ml
358
21.16562124
3924
5290
0.809844966


36
1-D7
AC-153
Guanabenz acetate
5051-62-7
231.1
2 mg/ml
12
21.6369888
4065
5531
0.809548771


304
4-G5
DL-144
Anastrozole 
120511-73-1
293.4
2 mg/ml 
104
17.04306387
3567
5290
0.806630684


56
1-F7
AC-194
Nisoxetine HCl
57754-86-6
271.4
2 mg/ml 
507
18.42554323
4059
5531
0.805484546


324
5-A5
DL-219
Cabergoline
81409-90-7
451.6
2 mg/ml 
278
11.07132593
3294
4930
0.801571305


573
8-B4
DL-522
Toltrazuril
69004-03-1
425.4
2 mg/ml 
697
11.75394259
3520
4361
0.800769364


471
6-H2
DL-429
Levamisole HCl
16595-80-5
204.3 
2 mg/ml
212
24.47432934
3833
4303
0.800367153


120
2-D11
AC-891
Levallorphan tartrate
71-82-9
283.4 
2 mg/ml
190
17.64183684
3532
5627
0.797215133


373
5-F4
DL-335
Azithromycin
83905-01-5
749.0
2 mg/ml
155
6.67553409
3632
4930
0.797151593


114
2-D5
AC-814
Amoxapine
14028-44-5
313.8
2 mg/ml
547
15.93424536
3721
5627
0.796965819


73
1-H4
DL-555
Zonisamide
68291-97-4
212.2 
2 mg/ml
756
23.55950585
3961
5531
0.793685158


222
3-G3
EI-107
Ketoconazole
65277-42-1
531.4
2 mg/ml 
82
9.408338565
3122
4352
0.792990814


279
4-D10
DL-289
Rofecoxib
162011-90-7
314.4 
2 mg/ml
633
15.90519334
3381
5290
0.790019508


378
5-F9
DL-340
Bromhexine HCl
611-75-6
376.1
2 mg/ml
518
13.2930324
3661
4930
0.783858348









Table 4-21 is a continuation of Table 4-20.


















TABLE 4-21







594
8-D5
NS-108
Dihydroergocristine
24730-10-7
611.7
2 mg/ml
586
8.17330653
3229
4361
0.774703516





mesylate










258
4-B9
DL-283
Guaiacol
90-05-1
124.1
2 mg/ml
249
40.27690858
3887
5290
0.772924515


551
7-H2
DL-505
Stanozolol
10418-03-8
328.5
2 mg/ml
648
15.22060795
3671
4940
0.770943044


539
7-F10
DL-494
Rimantadine HCl
1501-84-4
179.3
2 mg/ml
605
27.88500025
3608
4940
0.769597906


523
7-E4
DL-480
Progesterone
57-83-0
314.5
2 mg/ml
590
15.89966421
3990
4940
0.7684953


278
4-D9
DL-288
Rivastigmine
123441-03-2
250.3
2 mg/ml
626
19.97254654
3956
5290
0.753877214


13
1-B4
AC-105
Amiodarone
1951-25-3
645.3
2 mg/ml
96
7.748050986
3497
5531
0.752177709


163
3-A4
AR-107
Propranolol HCl s(−)
04199-10-4
259.4
2 mg/ml
596
19.27887224
3883
4352
0.749891664


168
3-A9
GR-103
Fenretinide
65646-68-6
391.6
2 mg/ml
482
12.76948693
209
4352
0.749416737


60
1-F11
AC-220
Pirenzepine 2HCl
28797-61-7
351.4
2 mg/ml
158
14.22833264
4106
5531
0.749389861


241
4-A2
EI-217
Tranylcypromine
1986-47-6
133.2
2 mg/ml
705
37.53902557
3535
5290
0.749206869


589
8-C10
NO-101
Naloxone HCl
357-08-4
327.4
2 mg/ml
486
15.27260749
3472
4361
0.746261252


596
8-D7
NS-140
Fluoxetine HCl
56296-78-7
309.3
2 mg/ml
228
16.16374207
2842
4361
0.743795192


103
2-C4
AC-765
Promethazine HCl
58-33-3
284.4
2 mg/ml
563
17.57924327
3585
5627
0.743703555


217
3-F8
D-108
Clothiapine
2058-52-8
343.9
2 mg/ml
19
14.53990252
3486
4352
0.741633402


62
1-G3
AC-222
Tropicamide
1508-75-4
284.4
2 mg/ml
714
17.58327395
4088
5531
0.739761701


246
4-A7
EI-287
Nimesulide
51803-78-2
308.3
2 mg/ml
129
16.21718101
3984
5290
0.735639128


299
4-F10
DL-172
Levetiracetam
102767-28-2
170.2
2 mg/ml
222
29.37496306
3985
5290
0.734915839


640
8-H11
DL-559
Benzydamine
642-72-8
309.4
2 mg/ml
173
16.15954461
2873
4361
0.732691877


337
5-B8
DL-197
Pramipexole
104632-26-0
211.3
2 mg/ml
172
23.65955714
4151
4930
0.729868388


478
6-H9
DL-436
Loratadine
79794-75-5
382.9
2 mg/ml
400
13.05845085
2345
4303
0.728053486


205
3-E6
CA-310
Trifluoperazine
440-17-5
407.5
2 mg/ml
708
12.26979623
256
4352
0.724331469


588
8-C9
NH-107
Levocabastine HCl
79547-78-7
420.5
2 mg/ml
224
11.88971208
3318
4361
0.722309799


130
2-E11
DL-247
Clemastine fumarate
14976-57-9
343.9
2 mg/ml
434
14.53909034
3387
5627
0.7187288


276
4-D7
GR-240
Pioglitazone
112529-15-4
356.4
2 mg/ml
162
14.02733675
4536
5290
0.717516371


419
6-B10
DL-204
Doxazosin mesylate
77883-43-3
451.5
2 mg/ml
439
11.07453561
3402
4303
0.717212737


54
1-F5
AC-192
Sotalol HCl
959-24-0
272.4
2 mg/ml
616
18.35746238
3974
5531
0.715704643


294
4-F5
KC-135
Tolazamide
1156-19-0
311.4
2 mg/ml
692
16.05622144
4477
5290
0.711525996


436
6-D7
DL-394
Fenbufen
36330-85-5
254.3
2 mg/ml
18
19.66273069
3926
4303
0.70843363









Table 4-22 is a continuation of Table 4-21.


















TABLE 4-22







262
4-C3
GR243
Raloxifene HCl
82640-04-8
473.6
2 mg/ml
609
10.55752851
1665
5290
0.708040409


325
5-A6
DL-207
Dilazep
35898-87-4
604.7
2 mg/ml
540
8.268511921
3015
4930
0.704993308


600
8-D11
NS-531
Mesoridazine besylate
32672-69-8
386.6
2 mg/ml
463
12.93386087
3463
4361
0.704082553


221
3-G2
DM200
Calcitriol
32222-06-3
416.6
2 mg/ml
285
12.00048204
899
4352
0.702353895


626
8-G7
DL-534
Bosentan
147536-97-8
551.6
2 mg/ml
219
9.064110964
3640
4361
0.699311155


413
6-B4
DL-374
Dextromethorphan HBr
125-69-9
271.4
2 mg/ml
301
18.42258122
3803
4303
0.699149884


251
4-B2
FR-111
Erogothioneine
497-30-3
229.3
2 mg/ml
194
21.80522872
3758
5290
0.694504259


169
3-A10
DL-269
Gefitinib
184475-35-2
446.9
2 mg/ml
378
11.18786033
589
4352
0.676746786


450
6-E11
DL-408
Formestane
566-48-3
302.4
2 mg/ml
260
16.53344731
3443
4303
0.675945227


274
4-D5
DL-284
Lapatinib
388082-78-8
581.1
2 mg/ml
165
8.604809775
2721
5290
0.674794404


107
2-C8
AC-807
Spiperone
749-02-0
395.5
2 mg/ml
636
12.64283336
4648
5627
0.673262474


24
1-C5
AC-124
Propafenone
54063-53-5
341.5
2 mg/ml
565
14.64325059
4080
5531
0.672731806


75
1-H6
AC-322
Naltrexone HCl
16676-29-2
341.4
2 mg/ml
46
14.6451219
4005
5531
0.669873202


411
6-B2
DL-373
Dehydroepiandrosterone
53-43-0
288.4
2 mg/ml
258
17.33499967
3541
4303
0.665805893


177
3-B8
DL-239
Efaroxan
89197-32-0
216.3
2 mg/ml
32
23.11761684
3978
4352
0.665437756


244
4-A5
EI-249
Troleandomycin
2751-09-9
814.0
2 mg/ml
713
6.142590358
4219
5290
0.649976365


216
3-F7
D-107
Clozapine
5786-21-0
326.8
2 mg/ml
26
15.29838287
3153
4352
0.64713495


16
1-B7
AC-110
Loperamide
34552-83-5
477.1
2 mg/ml
740
10.48104737
3896
5531
0.625278428


95
2-B6
AC-749
Chlorpromazine HCl
69-09-0
318.9
2 mg/ml
366
15.68030345
4084
5627
0.624779198


176
3-B7
CT-100
Fumagillone
23110-15-8
458.6
2 mg/ml
287
10.90377748
1296
4352
0.614764439


17
1-B8
AC-116
Fluspirilene
1841-19-6
475.6
2 mg/ml
236
10.51332972
2838
5531
0.580866948


164
3-A5
AR-112
Carvedilol
72656-09-3
406.5
2 mg/ml
338
12.30055208
518
4352
0.57524683


171
3-B2
DL-271
Imatinib
152459-95-5
493.6
2 mg/ml
402
10.12931653
3567
4352
0.564466886


199
3-D10
CA-216
Niguldipine HCl
113317-61-6
609.7
2 mg/ml
128
8.200368055
3173
4352
0.532465032


97
2-B8
AC-753
Fluphenazine 2HCl
146-56-5
437.5
2 mg/ml
232
11.42775594
4441
5627
0.495399649


55
1-F6
AR-111
Maprotiline HCl
10347-81-6
277.4
2 mg/ml
588
18.02366803
4164
5531
0.443894106


235
3-H6
EI-167
Methiothepin maleate
19728-88-2
356.6
2 mg/ml
67
14.02305525
1036
4352
0.439367416









INDUSTRIAL APPLICABILITY
Multi-color fluorescent reporters are useful tools to visualize patterns of alternative splicing in cultured cells and in living organisms at a single cell resolution. The multi-color reporters have been utilized to search for cis-elements and trans-acting factors involved in the regulation of alternative splicing, and to screen for chemical compounds affecting the splicing patterns. In the present invention, the present inventors describe how to construct fluorescent alternative splicing reporter mini-genes for a nematode Caenorhabditis elegans, cultured cells and mice. The mini-gene construction is based on site-directed recombination and various mini-genes can be easily constructed by assembling modular DNA fragments such as a promoter, tag protein cDNAs, a genomic fragment of interest, fluorescent protein cDNAs, and a 3′ cassette in separate vectors. The present inventors also described points to be considered in designing fluorescent alternative splicing reporters. The splicing reporter system can theoretically be applied to any other organisms.

## Claims

1. A method for detecting an alternative splicing of a specific gene in a mammalian multicellular organism, the method comprising:
(a) introducing into the multicellular organism a DNA construct such that at least two different reporter genes are inserted into the specific gene which undergoes the alternative splicing, wherein the at least two different reporter genes are connected in tandem at the 3′ side of the specific gene in a different reading frame in the DNA construct, and wherein the transcripts of the at least two different reporter genes are each connected in frame to a different exon which is included in at least two different mature mRNAs generated by alternative splicing of the specific gene, wherein the transcripts of the at least two different reporter genes are both present on each of the at least two different mature mRNAs and wherein each reporter gene is translated only when the exon to which it is connected is present on the mature mRNA; and
(b) detecting the alternative splicing of the specific gene in the multicellular organism by the expression of the reporter genes,
wherein the mammalian multicellular organism is a rodent, and
wherein the DNA construct comprises an N-terminal tag.

2. A method for testing whether or not a compound to be tested affects an alternative splicing of a specific gene, the method comprising:
(a) introducing into a mammalian multicellular organism a DNA construct such that at least two different reporter genes are inserted into the specific gene which undergoes the alternative splicing, wherein the at least two different reporter genes are connected in tandem at the 3′ side of the specific gene in a different reading frame in the DNA construct, and wherein the transcripts of the at least two different reporter genes are each connected in frame to a different exon which is included in at least two different mature mRNAs generated by alternative splicing of the specific gene, wherein the transcripts of the at least two different reporter genes are both present on each of the at least two different mature mRNAs and wherein each reporter gene is translated only when the exon to which it is connected is present on the mature mRNA;
(b) allowing the multicellular organism to contact the compound to be tested;
(c) detecting the alternative splicing of the specific gene in the multicellular organism by the expression of the reporter genes; and
(d) determining whether or not the expression of the reporter genes detected in the (c) has changed compared to the expression of the reporter genes in a control which is not allowed to contact the compound to be tested,
wherein the mammalian multicellular organism is a rodent, and
wherein the DNA construct comprises an N-terminal tag.

3. A method for identifying a gene region affecting an alternative splicing of a specific gene, the method comprising:
(a) introducing into a mammalian multicellular organism a DNA construct such that at least two different reporter genes are inserted into the specific gene which undergoes the alternative splicing, wherein the at least two different reporter genes are connected in tandem at the 3′ side of the specific gene in a different reading frame in the DNA construct, and wherein the transcripts of the at least two different reporter genes are each connected in frame to a different exon which is included in at least two different mature mRNAs generated by alternative splicing of the specific gene, wherein the transcripts of the at least two different reporter genes are both present on each of the at least two different mature mRNAs and wherein each reporter gene is translated only when the exon to which it is connected is present on the mature mRNA;
(b) treating the multicellular organism with a mutagen;
(c) detecting the alternative splicing of the specific gene in the multicellular organism by the expression of the reporter genes;
(d) selecting an individual in which the expression of the reporter genes detected in the (c) has changed compared to the expression of the reporter genes in a control which is not subjected to the mutagen treatment; and
(e) identifying a mutated gene region in the individual,
wherein the mammalian multicellular organism is a rodent, and
wherein the DNA construct comprises an N-terminal tag.

4. A method for identifying a region in a specific gene affecting an alternative splicing of the specific gene, the method comprising:
(a) introducing into a mammalian multicellular organism a DNA construct such that at least two different reporter genes are inserted into the specific gene into which a mutation has been introduced and which undergoes the alternative splicing, wherein the at least two different reporter genes are connected in tandem at the 3′ side of the specific gene in a different reading frame in the DNA construct, and wherein the transcripts of the at least two different reporter genes are each connected in frame to a different exon which is included in at least two different mature mRNAs generated by alternative splicing of the specific gene, wherein the transcripts of the at least two different reporter genes are both present on each of the at least two different mature mRNAs and wherein each reporter gene is translated only when the exon to which it is connected is present on the mature mRNA;
(b) detecting the alternative splicing of the specific gene in the multicellular organism by the expression of the reporter genes; and
(c) determining whether or not the expression of the reporter genes detected in the (b) has changed compared to the expression of the reporter genes in a control in which a mutation has not been introduced into the specific gene,
wherein the mammalian multicellular organism is a rodent, and
wherein the DNA construct comprises an N-terminal tag.

5. The method according to any one of claims 1 to 4, wherein each of the transcripts of the at least two different reporter genes is connected in frame to a mutually exclusive exon which is included in a different transcript as a result of the alternative splicing.

6. The method according to any one of claims 1 to 4, wherein the specific gene is linked to a promoter so that the specific gene functions under the control of the promoter, wherein the promoter is one of a tissue-specific promoter and a developmental stage-specific promoter.

7. The method according to any one of claims 1 to 4, wherein the multicellular organism is a mouse.

8. The method according to any one of claims 1 to 4, wherein the specific gene is fibroblast growth factor-receptor 2 (FGFR2).

9. The method according to any one of claims 1 to 4, wherein the at least two different reporter genes are each a gene encoding a fluorescent protein.

10. The method according to any one of claims 1 to 4, wherein the N-terminal tag comprises Glutathione S-transferase (GST).