Abstract:
Provided herein are methods and materials for identifying compounds that modulate polypyrimidine tract binding protein (PTB), a protein that functions as a negative regulator of pre-mRNA splicing by blocking the inclusion of numerous alternative exons into mRNA.

Description:
CROSS-RELATED APPLICATIONS 
       [0001]    The present application claims the benefit of the filing date of provisional application 61/118,845, filed on Dec. 1, 2008, which is incorporated by reference in its entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to using alternative splicing mechanisms to identify compounds that modulate the activity or expression of polypyrimidine-tract binding protein (PTB). 
       BACKGROUND 
       [0003]    Mammalian cells often utilize endogenous alternative splicing mechanisms, whereby multiple mRNAs from a single transcript may be produced. The spliced products may have very different, or even conflicting, functions. Alternative splicing may produce splice variants with differential functions, which may be critical for cellular development and/or homeostasis, and/or intra- and intercellular communication. In addition, modulating splicing regulation can result in consequences related to human disease, such as cancer. 
         [0004]    PTB is an RNA binding protein with multiple functions in the regulation of RNA processing and internal ribosomal entry site (IRES)-mediated translation. PTB was originally identified as a protein that bound to the pyrimidine-rich region within introns. PTB is a negative regulator of pre-mRNA splicing by blocking the inclusion of numerous alternative exons into mRNA. Accordingly, PTB expression and activity provides a nexus between disease and cellular mechanisms related to the removal of introns from mRNA precursors. 
       SUMMARY OF THE INVENTION 
       [0005]    Provided herein is a method for screening for a modulator of PTB. An identified modulator may inhibit or induce PTB activity. The method may comprise providing a cell that comprises PTB and a reporter system. The cell may be mammalian or non-mammalian. The PTB may be endogenously expressed and/or expressed from a heterologous nucleic acid. The heterologous nucleic acid may be a vector. The reporter system may comprise a first PTB target gene operably linked to a reporter sequence. A candidate modulator compound may be contacted with the cell, or the cell may be contacted with the modulator compound. The candidate modulator may be from a library of compounds. The library of compounds may be selected from the group consisting of a peptide library, a natural products library, a cDNA library, a combinatorial library, an oligosaccharide library, a drug library, phage display library, and a small molecule library. The compound may be expressed in the cell. The level of expression of the PTB target gene may be measured. A modulator of PTB may be identified by a change in expression of the PTB target as compared to a control. The first PTB target gene may be a minigene. The PTB target gene may encode a protein selected from the group consisting of c-src, α-actinin, FGF-R2, calcitonin/CGRP, GABA A γ2, α-tropomyosin, PTB1, PTB2, PTB4, GABBR1, INHBE, PICALM, GARNL1, MEIS1, NUMB, PPP3CB, and/or PTBP2. The minigene may encode a fragment of c-src, α-actinin, FGF-R2, calcitonin/CGRP, GABA A γ2, α-tropomyosin, PTB1, PTB2, PTB4, GABBR1, INHBE, PICALM, GARNL1, MEIS1, NUMB, PPP3CB, and/or PTBP2. The minigene may comprise exon 9 to exon 11 of PTBP2. The minigene may comprise exon 14 to exon 16 of GABBR1. The first gene or minigene may be operably linked to a reporter. The first minigene comprising exon 9 to exon 11 of PTBP2 may further comprise exon 11 operably linked to a first reporter. The first minigene comprising exon 14 to exon 16 of GABBR1 may further comprise exon 16 operably linked to a first reporter. 
         [0006]    The reporter system may further comprise a second PTB target gene or minigene. The second PTB target gene may encode a protein selected from the group consisting of c-src, α-actinin, FGF-R2, calcitonin/CGRP, GABA A γ2, α-tropomyosin, PTB1, PTB2, PTB4, GABBR1, INHBE, PICALM, GARNL1, MEIS1, NUMB, PPP3CB, and/or PTBP2. The second minigene may encode a fragment of c-src, α-actinin, FGF-R2, calcitonin/CGRP, GABA A γ2, α-tropomyosin, PTB1, PTB2, PTB4, GABBR1, INHBE, PICALM, GARNL1, MEIS1, NUMB, PPP3CB, and/or PTBP2. The second minigene may comprise exon 9 to exon 11 of PTBP2. The second minigene may comprise exon 14 to exon 16 of GABBR1. The second gene or minigene may be operably linked to a reporter. The second minigene comprising exon 9 to exon 11 of PTBP2 may further comprise exon 11 operably linked to a second reporter. The second minigene comprising exon 14 to exon 16 of GABBR1 may further comprise exon 16 operably linked to a second reporter. 
         [0007]    The first or second reporter may not be functionally expressed when exon 10 of PTBP2 or exon 15 of GABBR1 is skipped from the transcript of the first or second PTB target, respectively. The first or second reporter may be functionally expressed when exon 10 of PTBP2 or exon 15 of GABBR1 is spliced away from the transcript of the first or second PTB target, respectively. The first or second reporter may be functionally expressed when exon 10 of PTBP2 or exon 15 of GABBR1 is included in the transcript of the first or second PTB target, respectively. The first or second reporter may not be functionally expressed when exon 10 of PTBP2 or exon 15 of GABBR1 is included in the transcript of the first or second PTB target, respectively. 
         [0008]    The level of expression of a PTB target may be measured by the level of PTB target gene encoded mRNA. The level of PTB target gene encoded mRNA may be measured by RT-PCR. The level of expression may be measured by reporter output. The reporter output may be fluorescence. 
         [0009]    The control may be a cell. The control cell may comprise PTB and the reporter system. The cell may be contacted with a modulator compound that induces, or suppresses, or inhibits, or inhibits completely, PTB-expression and/or activity. The level(s) of expression and/or activity of PTB in the cell in contact with, or formerly in contact with, may be compared to the level(s) of expression and/or activity of PTB in the control cell. 
         [0010]    Also provided herein is a method for treating a subject diagnosed with a disease. The method may comprise administering the PTB modulator compound identified by the method described herein to a subject in need thereof. The disease may be cancer. The cancer may be ovarian cancer. The subject may be a mammal. The mammal may be a human. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0011]      FIG. 1  shows immunoblot analysis of PTB expression in cell lines. 
           [0012]      FIG. 2  shows the effects of PTB knockdown using a vector-based DOX (doxycycline)-inducible PTB siRNA. 
           [0013]      FIG. 3  shows a tumor growth curve in a xenograft mouse model. 
           [0014]      FIG. 4  shows validation of microarray analysis results related to altered expression and altered splicing pattern. 
           [0015]      FIG. 5  shows a schematic drawing of a reporter system for detection of PTB activity. 
           [0016]      FIG. 6  shows a schematic drawing of lentiviral vectors for use in a reporter system. 
           [0017]      FIG. 7  shows a schematic drawing of lentiviral vector expressing DOX-inducible siRNA. 
           [0018]      FIG. 8  shows diagram of changes in fluorescent intensity after PTB knockdown by DOX-induced PTB siRNA. 
           [0019]      FIG. 9  shows regulation of alternative splicing of exon 15 of GABBR1 by PTB. 
           [0020]      FIG. 10  shows a schematic drawing of a reporter system using alternative splicing of GABBR1. 
           [0021]      FIG. 11  shows detection of short and long-form SVs of GABBR1 by fluorescent proteins. 
       
    
    
     DETAILED DESCRIPTION 
       [0022]    The inventors have made the surprising discovery that altered expression and splicing activity of polypyrimidine-tract binding (PTB) protein may be directly related to disease. The ability to identify compounds that modulate PTB expression and/or activity may be useful as a therapeutic for treating a subject having a disease, or predisposed to a disease, as many diseases may be related to alterations in the regulation of splicing of PTB target nucleic acids. 
         [0023]    The methods and materials described herein use a PTB and PTB-targeted nucleic acid sequences in a reporter system to recapitulate a splicing pathway in a cell. The splicing pathway may be induced and compounds measured for their effect on PTB and its splicing-related activity. 
       1. DEFINITIONS 
       [0024]    The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. 
         [0025]    For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. 
         [0026]    The numbering of PTBP2 exons used herein corresponds to PTBP2 mRNA of accession number NM — 021190. The numbering of GABBR1 exons used herein is based on the GABBR1 mRNA of accession number NM — 001470. 
         [0027]    a. Fragment 
         [0028]    “Fragment” as used herein may mean a portion of a reference peptide or polypeptide or nucleic acid sequence. 
         [0029]    b. Identical 
         [0030]    “Identical” or “identity” as used herein in the context of two or more polypeptide or nucleotide sequences, may mean that the sequences have a specified percentage of residues or nucleotides that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. 
         [0031]    c. Operably Linked 
         [0032]    “Operably linked” as used herein may mean a functional linkage between two polynucleotides, for example a first polynucleotide and a second polynucleotide, wherein expression of one polynucleotide affects transcription and/or translation of the other polynucleotide. 
         [0033]    d. Skipped From 
         [0034]    “Skipped from” as used herein may mean “not included in” or “spliced away.” 
         [0035]    e. Substantially Complementary 
         [0036]    “Substantially complementary” as used herein may mean that a first sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the complement of a second sequence over a a region of 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 or more nucleotides or amino acids. Intermediate lengths may mean any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like. 
         [0037]    f. Substantially Identical 
         [0038]    “Substantially identical” as used herein may mean that a first and second nucleotide or amino acid sequence are at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over a region of 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 or more nucleotides or amino acids. Intermediate lengths may mean any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like. Substantially identical may also mean the first sequence nucleotide or amino acid sequence is substantially complementary to the complement of the second sequence. 
         [0039]    g. Variant 
         [0040]    “Variant” as used herein in the context of a nucleic acid may mean a substantially identical or substantially complementary sequence. A variant in reference to a nucleic acid may further mean a nucleic acid that may contain one or more substitutions, additions, deletions, insertions, or may be fragments thereof. A variant may also be a nucleic acid capable of hybridizing under moderately stringent conditions and specifically binding to a nucleic acid encoding the agent. Hybridization techniques are well known in the art and may be conducted under moderately stringent conditions. 
         [0041]    A variant in reference to a peptide may further mean differing from a native peptide in one or more substitutions, deletions, additions and/or insertions, or a sequence substantially identical to the native peptide sequence. The ability of a variant to react with antigen-specific antisera may be enhanced or unchanged, relative to the native protein, or may be diminished by less than 50%, or less than 20%, relative to the native peptide. Such variants may generally be identified by modifying one of the peptide sequences encoding an agent and evaluating the reactivity of the modified peptide with antigen-specific antibodies or antisera as described herein. Variants may include those in which one or more portions have been removed such as an N-terminal leader sequence or transmembrane domain. Other variants may include variants in which a small portion (e.g., 1-30 amino acids, or 5-15 amino acids) has been removed from the N- and/or C-terminal of the mature protein. 
         [0042]    A variant in reference to a peptide may contain conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry may expect the secondary structure and hydrophobic nature of the polypeptide to be substantially unchanged. Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also contain nonconservative changes. Variant peptides may differ from a native sequence by substitution, deletion or addition of amino acids. Variants may also be modified by deletion or addition of amino acids, which have minimal influence on the immunogenicity, secondary structure, hydropathic, and hydrophobic nature of the polypeptide. 
       2. METHOD OF IDENTIFYING MODULATORS OF PTB 
       [0043]    Provided herein is a method of screening for modulators of PTB. A cell may be provided that comprises PTB and a reporter system, which may comprise a first PTB target gene operably linked to a first reporter sequence. The reporter system may further comprise a second PTB target gene operably linked to a second reporter sequence. 
         [0044]    The cell may be contacted with a candidate modulator compound. After contact by the candidate compound, the level of expression of the PTB target gene may be measured. A modulator of PTB may be identified by a change in expression of the PTB target gene compared to a control. The level of expression of the PTB target gene may be measured as mRNA and/or protein. 
         [0045]    a. PTB 
         [0046]    Polypyrimidine-tract binding protein (PTB) may be any mammalian PTB protein as well as variants thereof. The mammalian PTB may be human. Representative examples of PTB include those shown in Table 1. 
         [0000]    
       
         
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Organism 
                 Gene and Accession No. 
               
               
                   
               
             
             
               
                 human 
                 PTBP1 isoform a (NCBI accession no. 
               
               
                   
                 NP_002810), 
               
               
                   
                 PTBP1 isoform b (NCBI accession no. 
               
               
                   
                 NP_114367), 
               
               
                   
                 PTBP1 isoform c (NCBI accession no. 
               
               
                   
                 NP_114366), and/or 
               
               
                   
                 PTBP1 isoform d (NCBI accession no. 
               
               
                   
                 NP_787041) 
               
               
                 
                   S. scrofa 
                 
                 PTB4, NCBI protein accession no. CAA63597 
               
               
                 
                   Mus musculis 
                 
                 PTB1, NCBI protein accession no. P17225 
               
               
                 
                   R. norvegicus 
                 
                 PTB4, NCBI protein accession no. Q00438 
               
               
                 
                   X. laevis 
                 
                 PTB4, NCBI protein accession no. AAF00041 
               
               
                 
                   D. melanogaster 
                 
                 PTB4, NCBI protein accession no. AAF22979 
               
               
                 
                   C. elegans 
                 
                 PTB4, NCBI protein accession no. T20381 
               
               
                 
                   Danio rerio 
                 
                 ptbp1, NCBI accession no. NP_001018313 
               
               
                 
                   Gallus gallus 
                 
                 PTBP1, NCBI accession no. NP_001026106 
               
               
                   Xenopus  ( Silurana ) 
                 ptbp1, NCBI aqccession no. NP_001011140 
               
               
                 
                   tropicalis 
                 
               
               
                 
                   Pan troglodytes 
                 
                 PTBP1, NCBI accession no. XP_001172084 
               
               
                 
                   Equus caballus 
                 
                 PTB1, NCBI accession no. XP_001915461 
               
               
                 
                   Macaca mulatto 
                 
                 PTBP1 isoform 1, NBI accession no. 
               
               
                   
                 XP_001092088 
               
               
                 
                   Canis lupus familiaris 
                 
                 PTBP1, NCBI accession no. XP_868639 
               
               
                 
                   Bos taurus 
                 
                 PTBP1, NCBI accession no. NP_776867 
               
               
                   
               
             
          
         
       
     
         [0047]    PTB may be expressed from a cell chromosome and/or a heterologous nucleic acid. The heterologous nucleic acid may be a vector or plasmid. PTB expression is typically directed by a promoter. A promoter can be naturally associated with a nucleic acid sequence, as can be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment and/or exon. Alternatively, the promoter can be from a different gene, or from a gene from a different species of organism (“heterologous”). Expression of PTB may be controlled by an inducible promoter, such as, for example, Gal1-10, Gal1, GalL, GalS, or CUP1 or a repressible promoter, such as Met25, for expression in yeast. An inducible promoter may be active under environmental or developmental regulation. The inducible promoter may be capable of functioning in a eukaryotic host organism. These promoters include naturally occurring yeast and mammalian inducible promoters as well as synthetic promoters designed to function in a eukaryotic host. An important functional characteristic of an inducible promoter is its inducibility by exposure to an environmental inducing agent. Appropriate environmental inducing agents include exposure to heat, various steroidal compounds, divalent cations (including Cu +2  and Zn +2 ), galactose, tetracycline, IPTG (isopropyl-β-D thiogalactoside), as well as other naturally occurring and synthetic inducing agents and gratuitous inducers. The inducible promoter may be a vector-based DOX (doxycycline)-inducible promoter. Synthetic inducible promoter systems are also available for use. Suitable expression cassettes are readily available for heterologous expression in many different eukaryotic cells including various yeast species and mammalian cells. PTB nucleic acids can include at least one termination signal and/or polyadenylation signal, as needed. 
         [0048]    b. Reporter System 
         [0049]    (1) PTB Target Gene 
         [0050]    The reporter system may comprise one or more PTB target genes. For example, there may be at least 1, 2, 3, 4, 5, 6, 7, or more target genes. Any of the PTB target genes may be expressed from a heterologous nucleic acid. The heterologous nucleic acid may be a vector or plasmid. The PTB target genes may be expressed from two or more separate vectors or plasmids. The one or more PTB target genes may be nucleotide sequences. The nucleotide sequences may be full-length pre-mRNA nucleotide sequences of the PTB target or variants thereof. The one or more PTB target genes may be pre-mRNA sequences encoded by one or more genes. The PTB target pre-mRNA sequence may be spliced via a PTB-driven mechanism. The gene may encode c-src, α-actinin, FGF-R2, calcitonin/CGRP, GABA A γ2, α-tropomyosin, PTB1, PTB2, PTB4, GABBR1, INHBE, PICALM, GARNL1, MEIS1, NUMB, PPP3CB, and/or PTBP2. The gene may be one or more genes selected from Table 2. 
         [0051]    The one or more PTB target genes may be pre-mRNA sequences encoded by one or more minigenes. The PTB target pre-mRNA sequence may be spliced via a PTB-driven mechanism. The minigene may be a fragment of a gene or a variant thereof. Any of the PTB target minigenes may be expressed from a heterologous nucleic acid. The heterologous nucleic acid may be a vector or plasmid. The PTB target minigenes may be expressed from two or more separate vectors or plasmids. A minigene may comprise a genomic sequence spanning one or more, two or more, three or more, four or more, or five or more exons of the PTB target. A minigene may comprise a genomic sequence spanning one or more, two or more, three or more, four or more, or five or more introns of a PTB target gene. A minigene may comprise a genomic sequence comprising any number of introns or exons of a PTB target. The minigene may comprise a genomic sequence comprising exons 1 through 3, 2 through 4, 4 through 6, 5 through 7, 6 through 8, 7 through 9, 8 through 10, 9 through 11, 10 through 12, 11 through 13, and/or 12 through 14 of PTBP2. The minigene may comprise a genomic sequence comprising exons 1 through 3, 2 through 4, 4 through 6, 5 through 7, 6 through 8, 7 through 9, 8 through 10, 9 through 11, 10 through 12, 13 through 15, 14 through 16, 15 through 17, 16 through 18, 17 through 19, 18 through 20, 19 through 21, 20 through 22, and/or 21 through 23 of GABBR1. The minigene may encode a fragment of c-src, α-actinin, FGF-R2, calcitonin/CGRP, GABA A γ2, α-tropomyosin, PTB1, PTB2, PTB4, GABBR1, INHBE, PICALM, GARNL1, MEIS1, NUMB, PPP3CB, and/or PTBP2. The minigene may encode a fragment of one or more proteins encoded by the genes selected from Table 2. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 PTB-regulated genes 
               
             
          
           
               
                 GENE_SYMBOL 
                 Gene Name 
               
               
                   
               
               
                 STX3A 
                 syntaxin 3a 
               
               
                 CSDE1 
                 cold shock domain containing e1, rna-binding 
               
               
                 CCNB1IP1 
                 cyclin b1 interacting protein 1 
               
               
                 ANKRD10 
                 ankyrin repeat domain 10 
               
               
                 TSC22D3 
                 tsc22 domain family, member 3 
               
               
                 PPP3CB 
                 protein phosphatase 3 (formerly 2b), catalytic 
               
               
                   
                 subunit, beta isoform (calcineurin a beta) 
               
               
                 EXOC7 
                 exocyst complex component 7 
               
               
                 EXO1 
                 exonuclease 1 
               
               
                 TDRD3 
                 tudor domain containing 3 
               
               
                 TPM2 
                 tropomyosin 2 (beta) 
               
               
                 LOC55565 
                 hypothetical protein loc55565 
               
               
                 SEC13L1 
                 sec13-like 1 ( s. cerevisiae ) 
               
               
                 TMEM16C 
                 transmembrane protein 16c 
               
               
                 NFYC 
                 nuclear transcription factor y, gamma 
               
               
                 EHBP1 
                 eh domain binding protein 1 
               
               
                 MLLT10 
                 myeloid/lymphoid or mixed-lineage leukemia 
               
               
                   
                 (trithorax homolog,  drosophila ); translocated 
               
               
                   
                 to, 10 
               
               
                 TSC2 
                 tuberous sclerosis 2 
               
               
                 USP54 
                 ubiquitin specific peptidase 54 
               
               
                 CBS 
                 cystathionine-beta-synthase 
               
               
                 PCDH17 
                 protocadherin 17 
               
               
                 ARRB1 
                 arrestin, beta 1 
               
               
                 LRRFIP2 
                 leucine rich repeat (in flii) interacting protein 2 
               
               
                 CDK5RAP2 
                 cdk5 regulatory subunit associated protein 2 
               
               
                 BMPR2 
                 bone morphogenetic protein receptor, type ii 
               
               
                   
                 (serine/threonine kinase) 
               
               
                 KCNK2 
                 potassium channel, subfamily k, member 2 
               
               
                 KPNA1 
                 karyopherin alpha 1 (importin alpha 5) 
               
               
                 ABI2 
                 abl interactor 2 
               
               
                 SCARB1 
                 scavenger receptor class b, member 1 
               
               
                 AMT 
                 aminomethyltransferase (glycine cleavage 
               
               
                   
                 system protein t) 
               
               
                 GOLPH4 
                 golgi phosphoprotein 4 
               
               
                 MYO5A 
                 myosin va (heavy polypeptide 12, myoxin) 
               
               
                 C14orf118 
                 chromosome 14 open reading frame 118 
               
               
                 TPM1 
                 tropomyosin 1 (alpha) 
               
               
                 RNPC2 
                 rna-binding region (rnp1, rrm) containing 2 
               
               
                 NCOA7 
                 nuclear receptor coactivator 7 
               
               
                 PPP3CA 
                 protein phosphatase 3 (formerly 2b), catalytic 
               
               
                   
                 subunit, alpha isoform (calcineurin a alpha) 
               
               
                 USP16 
                 ubiquitin specific peptidase 16 
               
               
                 SCP2 
                 sterol carrier protein 2 
               
               
                 CATSPER2 
                 cation channel, sperm associated 2 
               
               
                 MCM7 
                 mcm7 minichromosome maintenance deficient 
               
               
                   
                 7 ( s. cerevisiae ) 
               
               
                 NYREN18 
                 nedd8 ultimate buster-1 
               
               
                 OCRL 
                 oculocerebrorenal syndrome of lowe 
               
               
                 OGT 
                 o-linked n-acetylglucosamine (glcnac) 
               
               
                   
                 transferase (udp-n- 
               
               
                   
                 acetylglucosamine:polypeptide-n- 
               
               
                   
                 acetylglucosaminyl transferase) 
               
               
                 NEDD4L 
                 neural precursor cell expressed, 
               
               
                   
                 developmentally down-regulated 4-like 
               
               
                 STOX2 
                 storkhead box 2 
               
               
                 SFRS2 
                 splicing factor, arginine/serine-rich 2 
               
               
                 NFATC4 
                 nuclear factor of activated t-cells, cytoplasmic, 
               
               
                   
                 calcineurin-dependent 4 
               
               
                 HMGCL 
                 3-hydroxymethyl-3-methylglutaryl-coenzyme 
               
               
                   
                 a lyase (hydroxymethylglutaricaciduria) 
               
               
                 39331 
                 septin 6 
               
               
                 SH3KBP1 
                 sh3-domain kinase binding protein 1 
               
               
                 ATP9B 
                 atpase, class ii, type 9b 
               
               
                 SYNE1 
                 spectrin repeat containing, nuclear envelope 1 
               
               
                 SMG7 
                 smg-7 homolog, nonsense mediated mrna 
               
               
                   
                 decay factor ( c. elegans ) 
               
               
                 SEPP1 
                 selenoprotein p, plasma, 1 
               
               
                 PICALM 
                 phosphatidylinositol binding clathrin assembly 
               
               
                   
                 protein 
               
               
                 PTPN13 
                 protein tyrosine phosphatase, non-receptor type 
               
               
                   
                 13 (apo-1/cd95 (fas)-associated phosphatase) 
               
               
                 CDC42BPA 
                 cdc42 binding protein kinase alpha (dmpk-like) 
               
               
                 ATP11C 
                 atpase, class vi, type 11c 
               
               
                 CTNNB1 
                 catenin (cadherin-associated protein), beta 1, 
               
               
                   
                 88 kda 
               
               
                 TSPAN1 
                 tetraspanin 1 
               
               
                 C20orf96 
                 chromosome 20 open reading frame 96 
               
               
                 PPA2 
                 pyrophosphatase (inorganic) 2 
               
               
                 EPB41 
                 erythrocyte membrane protein band 4.1 
               
               
                   
                 (elliptocytosis 1, rh-linked) 
               
               
                 ARHGEF7 
                 rho guanine nucleotide exchange factor (gef) 7 
               
               
                 DPYD 
                 dihydropyrimidine dehydrogenase 
               
               
                 DZIP1 
                 daz interacting protein 1 
               
               
                 MCM10 
                 mcm10 minichromosome maintenance 
               
               
                   
                 deficient 10 ( s. cerevisiae ) 
               
               
                 ISL1 
                 isl1 transcription factor, lim/homeodomain, 
               
               
                   
                 (islet-1) 
               
               
                 WDR45L 
                 wdr45-like 
               
               
                 POU1F1 
                 pou domain, class 1, transcription factor 1 
               
               
                   
                 (pit1, growth hormone factor 1) 
               
               
                 GDPD1 
                 glycerophosphodiester phosphodiesterase 
               
               
                   
                 domain containing 1 
               
               
                 LMAN2L 
                 lectin, mannose-binding 2-like 
               
               
                 FLJ10357 
                 hypothetical protein flj10357 
               
               
                 DDIT3 
                 dna-damage-inducible transcript 3 
               
               
                 NOX4 
                 nadph oxidase 4 
               
               
                 TXNIP 
                 thioredoxin interacting protein 
               
               
                 GABBR1 
                 gamma-aminobutyric acid (gaba) b receptor, 1 
               
               
                 LOC153222 
                 adult retina protein 
               
               
                 LETMD1 
                 letm1 domain containing 1 
               
               
                 LRRFIP1 
                 leucine rich repeat (in flii) interacting protein 1 
               
               
                 MEF2D 
                 mads box transcription enhancer factor 2, 
               
               
                   
                 polypeptide d (myocyte enhancer factor 2d) 
               
               
                 UROD 
                 uroporphyrinogen decarboxylase 
               
               
                 NUMB 
                 numb homolog ( drosophila ) 
               
               
                 AK2 
                 adenylate kinase 2 
               
               
                 SWAP70 
                 swap-70 protein 
               
               
                 RAPGEF2 
                 rap guanine nucleotide exchange factor (gef) 2 
               
               
                 TTLL6 
                 tubulin tyrosine ligase-like family, member 6 
               
               
                 CTSC 
                 cathepsin c 
               
               
                 RSN 
                 restin (reed-steinberg cell-expressed 
               
               
                   
                 intermediate filament-associated protein) 
               
               
                 MAPK10 
                 mitogen-activated protein kinase 10 
               
               
                 ZDHHC6 
                 zinc finger, dhhc-type containing 6 
               
               
                 KNS2 
                 kinesin 2 
               
               
                 TEX9 
                 testis expressed sequence 9 
               
               
                 MEF2A 
                 mads box transcription enhancer factor 2, 
               
               
                   
                 polypeptide a (myocyte enhancer factor 2a) 
               
               
                 TPM3 
                 tropomyosin 3 
               
               
                 PIK3C3 
                 phosphoinositide-3-kinase, class 3 
               
               
                 CSTA 
                 cystatin a (stefin a) 
               
               
                 OCA2 
                 oculocutaneous albinism ii (pink-eye dilution 
               
               
                   
                 homolog, mouse) 
               
               
                 BAG1 
                 bcl2-associated athanogene 
               
               
                 RPS24 
                 ribosomal protein s24 
               
               
                 HPS4 
                 le protein 
               
               
                 CTH 
                 cystathionase (cystathionine gamma-lyase) 
               
               
                 KIFAP3 
                 kinesin-associated protein 3 
               
               
                 YPEL5 
                 yippee-like 5 ( drosophila ) 
               
               
                 EXOSC3 
                 exosome component 3 
               
               
                 SSFA2 
                 sperm specific antigen 2 
               
               
                 CLTC 
                 clathrin, heavy polypeptide (hc) 
               
               
                 ERBB2IP 
                 erbb2 interacting protein 
               
               
                 C20orf19 
                 chromosome 20 open reading frame 19 
               
               
                 MERTK 
                 c-mer proto-oncogene tyrosine kinase 
               
               
                 WDR6 
                 wd repeat domain 6 
               
               
                 ANGEL2 
                 angel homolog 2 ( drosophila ) 
               
               
                 CREB3L1 
                 camp responsive element binding protein 3- 
               
               
                   
                 like 1 
               
               
                 CARM1 
                 coactivator-associated arginine 
               
               
                   
                 methyltransferase 1 
               
               
                 WDR25 
                 wd repeat domain 25 
               
               
                 TSLP 
                 thymic stromal lymphopoietin 
               
               
                 CALD1 
                 caldesmon 1 
               
               
                 C2orf13 
                 chromosome 2 open reading frame 13 
               
               
                 RECK 
                 reversion-inducing-cysteine-rich protein with 
               
               
                   
                 kazal motifs 
               
               
                 COMMD4 
                 comm domain containing 4 
               
               
                 ABLIM1 
                 actin binding lim protein 1 
               
               
                 CARS 
                 cysteinyl-trna synthetase 
               
               
                 PHF14 
                 phd finger protein 14 
               
               
                 ATXN7 
                 ataxin 7 
               
               
                 PTBP2 
                 polypyrimidine tract binding protein 2 
               
               
                 DDIT4 
                 dna-damage-inducible transcript 4 
               
               
                 PRSS16 
                 protease, serine, 16 ( thymus ) 
               
               
                 COPS5 
                 cop9 constitutive photomorphogenic homolog 
               
               
                   
                 subunit 5 ( arabidopsis ) 
               
               
                 ASPH 
                 aspartate beta-hydroxylase 
               
               
                 CCT4 
                 chaperonin containing tcp1, subunit 4 (delta) 
               
               
                 PRSS7 
                 protease, serine, 7 (enterokinase) 
               
               
                 PIGX 
                 phosphatidylinositol glycan, class x 
               
               
                 COG3 
                 component of oligomeric golgi complex 3 
               
               
                 LASS5 
                 lag1 longevity assurance homolog 5 
               
               
                   
                 ( s. cerevisiae ) 
               
               
                 IL21R 
                 interleukin 21 receptor 
               
               
                 B4GALT4 
                 udp-gal:betaglcnac beta 1,4- 
               
               
                   
                 galactosyltransferase, polypeptide 4 
               
               
                 TRERF1 
                 transcriptional regulating factor 1 
               
               
                 RAPH1 
                 ras association (ralgds/af-6) and pleckstrin 
               
               
                   
                 homology domains 1 
               
               
                 LYK5 
                 protein kinase lyk5 
               
               
                 MPZL1 
                 myelin protein zero-like 1 
               
               
                 USP28 
                 ubiquitin specific peptidase 28 
               
               
                 KIAA1109 
                 kiaa1371 protein 
               
               
                 VMD2 
                 vitelliform macular dystrophy 2 (best disease, 
               
               
                   
                 bestrophin) 
               
               
                 ROBO3 
                 roundabout, axon guidance receptor, homolog 
               
               
                   
                 3 ( drosophila ) 
               
               
                 SMNDC1 
                 survival motor neuron domain containing 1 
               
               
                 DLD 
                 dihydrolipoamide dehydrogenase (e3 
               
               
                   
                 component of pyruvate dehydrogenase 
               
               
                   
                 complex, 2-oxo-glutarate complex, branched 
               
               
                   
                 chain keto acid dehydrogenase complex) 
               
               
                 CRYZ 
                 crystallin, zeta (quinone reductase) 
               
               
                 IQGAP1 
                 iq motif containing gtpase activating protein 1 
               
               
                 SLC37A4 
                 solute carrier family 37 (glycerol-6-phosphate 
               
               
                   
                 transporter), member 4 
               
               
                 TNRC6A 
                 trinucleotide repeat containing 6a 
               
               
                 PLEKHH2 
                 pleckstrin homology domain containing, 
               
               
                   
                 family h (with myth4 domain) member 2 
               
               
                 PLD1 
                 phospholipase d1, phosphatidylcholine-specific 
               
               
                 PKP4 
                 plakophilin 4 
               
               
                 ARHGEF2 
                 rho/rac guanine nucleotide exchange factor 
               
               
                   
                 (gef) 2 
               
               
                 ITPA 
                 inosine triphosphatase (nucleoside triphosphate 
               
               
                   
                 pyrophosphatase) 
               
               
                 C3orf17 
                 chromosome 3 open reading frame 17 
               
               
                 CBLL1 
                 cas-br-m (murine) ecotropic retroviral 
               
               
                   
                 transforming sequence-like 1 
               
               
                 THOC5 
                 tho complex 5 
               
               
                 LOC81691 
                 exonuclease nef-sp 
               
               
                 AP1GBP1 
                 ap1 gamma subunit binding protein 1 
               
               
                 ADD1 
                 adducin 1 (alpha) 
               
               
                 PTBP1 
                 polypyrimidine tract binding protein 1 
               
               
                 TCF7 
                 transcription factor 7 (t-cell specific, hmg-box) 
               
               
                 NIPBL 
                 nipped-b homolog ( drosophila ) 
               
               
                 PFN2 
                 profilin 2 
               
               
                 CLUAP1 
                 clusterin associated protein 1 
               
               
                 ME1 
                 malic enzyme 1, nadp(+)-dependent, cytosolic 
               
               
                 JDP2 
                 jun dimerization protein 2 
               
               
                 SVIL 
                 supervillin 
               
               
                 ERO1LB 
                 ero1-like beta ( s. cerevisiae ) 
               
               
                 MDS025 
                 hypothetical protein mds025 
               
               
                 DHRS2 
                 dehydrogenase/reductase (sdr family) member 2 
               
               
                 DTX2 
                 kiaa1528 protein 
               
               
                 NRG4 
                 neuregulin 4 
               
               
                 FANCA 
                 fanconi anemia, complementation group a 
               
               
                 NOL5A 
                 nucleolar protein 5a (56 kda with kke/d repeat) 
               
               
                 ATP6V1H 
                 atpase, h+ transporting, lysosomal 50/57 kda, 
               
               
                   
                 v1 subunit h 
               
               
                 ACTN1 
                 actinin, alpha 1 
               
               
                 CRSP3 
                 cofactor required for sp1 transcriptional 
               
               
                   
                 activation, subunit 3, 130 kda 
               
               
                 SEC61A2 
                 sec61 alpha 2 subunit ( s. cerevisiae ) 
               
               
                 AZIN1 
                 antizyme inhibitor 1 
               
               
                 MRPS22 
                 mitochondrial ribosomal protein s22 
               
               
                 ITGA5 
                 integrin, alpha 5 (fibronectin receptor, alpha 
               
               
                   
                 polypeptide) 
               
               
                 MAP4 
                 microtubule-associated protein 4 
               
               
                 DCDC2 
                 doublecortin domain containing 2 
               
               
                 GFPT1 
                 glutamine-fructose-6-phosphate transaminase 1 
               
               
                 GMPR2 
                 guanosine monophosphate reductase 2 
               
               
                 ADHFE1 
                 alcohol dehydrogenase, iron containing, 1 
               
               
                 INHBE 
                 inhibin, beta e 
               
               
                 EPB41L1 
                 erythrocyte membrane protein band 4.1-like 1 
               
               
                 MAP3K4 
                 mitogen-activated protein kinase kinase kinase 4 
               
               
                 ACSM3 
                 acyl-coa synthetase medium-chain family 
               
               
                   
                 member 3 
               
               
                 ECHDC1 
                 enoyl coenzyme a hydratase domain containing 1 
               
               
                 TTC13 
                 tetratricopeptide repeat domain 13 
               
               
                 KLHDC1 
                 kelch domain containing 1 
               
               
                 KIAA1377 
                 kiaa1377 
               
               
                 UBE2V2 
                 ubiquitin-conjugating enzyme e2 variant 2 
               
               
                 ASS 
                 argininosuccinate synthetase 
               
               
                 SLC38A6 
                 solute carrier family 38, member 6 
               
               
                 MRPS27 
                 mitochondrial ribosomal protein s27 
               
               
                 TTC9C 
                 tetratricopeptide repeat domain 9c 
               
               
                 NEK7 
                 nima (never in mitosis gene a)-related kinase 7 
               
               
                 EAF1 
                 ell associated factor 1 
               
               
                 ITGB3BP 
                 integrin beta 3 binding protein (beta3- 
               
               
                   
                 endonexin) 
               
               
                 TRIM2 
                 tripartite motif-containing 2 
               
               
                 FNDC6 
                 fibronectin type iii domain containing 6 
               
               
                 C21orf6 
                 chromosome 21 open reading frame 6 
               
               
                 SFRS14 
                 splicing factor, arginine/serine-rich 14 
               
               
                 CBX5 
                 chromobox homolog 5 (hp1 alpha homolog, 
               
               
                   
                 drosophila) 
               
               
                 ATP1B3 
                 atpase, na+/k+ transporting, beta 3 polypeptide 
               
               
                 MOV10 
                 mov10, moloney leukemia virus 10, homolog 
               
               
                   
                 (mouse) 
               
               
                 PRR3 
                 proline rich 3 
               
               
                 SLC7A11 
                 solute carrier family 7, (cationic amino acid 
               
               
                   
                 transporter, y+ system) member 11 
               
               
                 MRPL20 
                 mitochondrial ribosomal protein 120 
               
               
                 PBX1 
                 pre-b-cell leukemia transcription factor 1 
               
               
                 CASP2 
                 caspase 2, apoptosis-related cysteine peptidase 
               
               
                   
                 (neural precursor cell expressed, 
               
               
                   
                 developmentally down-regulated 2) 
               
               
                 ANK3 
                 ankyrin 3, node of ranvier (ankyrin g) 
               
               
                 CYP4V2 
                 cytochrome p450, family 4, subfamily v, 
               
               
                   
                 polypeptide 2 
               
               
                 CTAGE5 
                 ctage family, member 5 
               
               
                 PDLIM7 
                 pdz and lim domain 7 (enigma) 
               
               
                 STRA6 
                 stimulated by retinoic acid gene 6 homolog 
               
               
                   
                 (mouse) 
               
               
                 IKIP 
                 ikk interacting protein 
               
               
                 CCPG1 
                 cell cycle progression 1 
               
               
                 FBXO38 
                 hypothetical protein flj13962 
               
               
                 PRKX 
                 protein kinase, x-linked 
               
               
                 TNIK 
                 traf2 and nck interacting kinase 
               
               
                 ASNS 
                 asparagine synthetase 
               
               
                 RBM25 
                 rna binding motif protein 25 
               
               
                 HIST1H2BD 
                 histone 1, h2bd 
               
               
                 KIAA0528 
                 kiaa0528 
               
               
                 SFRS10 
                 splicing factor, arginine/serine-rich 10 
               
               
                   
                 (transformer 2 homolog,  drosophila ) 
               
               
                 PTPDC1 
                 protein tyrosine phosphatase domain 
               
               
                   
                 containing 1 
               
               
                 COL5A2 
                 collagen, type v, alpha 2 
               
               
                 AGA 
                 aspartylglucosaminidase 
               
               
                 SFRS3 
                 splicing factor, arginine/serine-rich 3 
               
               
                 HDAC6 
                 histone deacetylase 6 
               
               
                 BTG1 
                 b-cell translocation gene 1, anti-proliferative 
               
               
                 SNX14 
                 sorting nexin 14 
               
               
                 ARFIP1 
                 adp-ribosylation factor interacting protein 1 
               
               
                   
                 (arfaptin 1) 
               
               
                 XBP1 
                 x-box binding protein 1 
               
               
                 PPFIA1 
                 protein tyrosine phosphatase, receptor type, f 
               
               
                   
                 polypeptide (ptprf), interacting protein (liprin), 
               
               
                   
                 alpha 1 
               
               
                 IFRD1 
                 interferon-related developmental regulator 1 
               
               
                 SNAP23 
                 synaptosomal-associated protein, 23 kda 
               
               
                 SH3YL1 
                 sh3 domain containing, ysc84-like 1 
               
               
                   
                 ( s. cerevisiae ) 
               
               
                 CAPZB 
                 capping protein (actin filament) muscle z-line, 
               
               
                   
                 beta 
               
               
                 CSPG5 
                 chondroitin sulfate proteoglycan 5 
               
               
                   
                 (neuroglycan c) 
               
               
                 LAS1L 
                 las1-like ( s. cerevisiae ) 
               
               
                 CCNA2 
                 cyclin a2 
               
               
                 GDAP1L1 
                 ganglioside-induced differentiation-associated 
               
               
                   
                 protein 1-like 1 
               
               
                 DCTN1 
                 dynactin 1 (p150, glued homolog,  drosophila ) 
               
               
                 HIPK3 
                 homeodomain interacting protein kinase 3 
               
               
                 NUP62 
                 nucleoporin 62 kda 
               
               
                 NECAP1 
                 necap endocytosis associated 1 
               
               
                 NDUFV3 
                 nadh dehydrogenase (ubiquinone) flavoprotein 
               
               
                   
                 3, 10 kda 
               
               
                 SLCO4A1 
                 solute carrier organic anion transporter family, 
               
               
                   
                 member 4a1 
               
               
                 CAMKK2 
                 calcium/calmodulin-dependent protein kinase 
               
               
                   
                 kinase 2, beta 
               
               
                 DLG2 
                 discs, large homolog 2, chapsyn-110 
               
               
                   
                 ( drosophila ) 
               
               
                 TBC1D23 
                 tbc1 domain family, member 23 
               
               
                 SNX13 
                 sorting nexin 13 
               
               
                 ATP2B4 
                 atpase, ca++ transporting, plasma membrane 4 
               
               
                 AURKB 
                 aurora kinase b 
               
               
                 CDC25A 
                 cell division cycle 25a 
               
               
                 FBXO25 
                 f-box protein 25 
               
               
                 SAP18 
                 sin3a-associated protein, 18 kda 
               
               
                 MMP10 
                 matrix metallopeptidase 10 (stromelysin 2) 
               
               
                 TRAPPC4 
                 trafficking protein particle complex 4 
               
               
                 ZNF195 
                 zinc finger protein 195 
               
               
                 KTN1 
                 kinectin 1 (kinesin receptor) 
               
               
                 CPEB1 
                 cytoplasmic polyadenylation element binding 
               
               
                   
                 protein 1 
               
               
                 ODF2 
                 outer dense fiber of sperm tails 2 
               
               
                 EFEMP2 
                 fibulin 4 
               
               
                 PCM1 
                 pericentriolar material 1 
               
               
                 KIAA0494 
                 kiaa0494 
               
               
                 DNAJC3 
                 dnaj (hsp40) homolog, subfamily c, member 3 
               
               
                 FEZ2 
                 fasciculation and elongation protein zeta 2 
               
               
                   
                 (zygin ii) 
               
               
                 SRRM1 
                 serine/arginine repetitive matrix 1 
               
               
                 TFRC 
                 transferrin receptor (p90, cd71) 
               
               
                 KIFC1 
                 kinesin family member c1 
               
               
                 OSBPL9 
                 hypothetical protein flj12492 
               
               
                 ARHGEF11 
                 rho guanine nucleotide exchange factor (gef) 
               
               
                   
                 11 
               
               
                 ZMYND12 
                 zinc finger, mynd-type containing 12 
               
               
                 MEIS1 
                 meis1, myeloid ecotropic viral integration site 
               
               
                   
                 1 homolog (mouse) 
               
               
                 FUSIP1 
                 fus interacting protein (serine/arginine-rich) 1 
               
               
                 CALCRL 
                 calcitonin receptor-like 
               
               
                 LEPRE1 
                 leucine proline-enriched proteoglycan 
               
               
                   
                 (leprecan) 1 
               
               
                 VLDLR 
                 very low density lipoprotein receptor 
               
               
                 APP 
                 amyloid beta (a4) precursor protein (peptidase 
               
               
                   
                 nexin-ii, alzheimer disease) 
               
               
                 FGG 
                 fibrinogen gamma chain 
               
               
                 TUG1 
                 taurine upregulated gene 1 
               
               
                 S100A4 
                 s100 calcium binding protein a4 (calcium 
               
               
                   
                 protein, calvasculin, metastasin, murine 
               
               
                   
                 placental homolog) 
               
               
                 NDUFS7 
                 nadh dehydrogenase (ubiquinone) fe—s protein 
               
               
                   
                 7, 20 kda (nadh-coenzyme q reductase) 
               
               
                 ATF3 
                 activating transcription factor 3 
               
               
                 LPHN2 
                 latrophilin 2 
               
               
                 SLC25A36 
                 solute carrier family 25, member 36 
               
               
                 TCF20 
                 transcription factor 20 (ar1) 
               
               
                 ASF1B 
                 asf1 anti-silencing function 1 homolog b 
               
               
                   
                 ( s. cerevisiae ) 
               
               
                 ART3 
                 adp-ribosyltransferase 3 
               
               
                 PACSIN2 
                 protein kinase c and casein kinase substrate in 
               
               
                   
                 neurons 2 
               
               
                 C6orf142 
                 chromosome 6 open reading frame 142 
               
               
                 ORAOV1 
                 oral cancer overexpressed 1 
               
               
                 TCF7L2 
                 transcription factor 7-like 2 (t-cell specific, 
               
               
                   
                 hmg-box) 
               
               
                 SLC1A4 
                 solute carrier family 1 (glutamate/neutral 
               
               
                   
                 amino acid transporter), member 4 
               
               
                 SEMA3C 
                 sema domain, immunoglobulin domain (ig), 
               
               
                   
                 short basic domain, secreted, (semaphorin) 3c 
               
               
                 WARS 
                 interferon-induced protein 53 
               
               
                 ATP2A2 
                 atpase, ca++ transporting, cardiac muscle, slow 
               
               
                   
                 twitch 2 
               
               
                 SLC16A6 
                 solute carrier family 16 (monocarboxylic acid 
               
               
                   
                 transporters), member 6 
               
               
                 C1orf91 
                 chromosome 1 open reading frame 91 
               
               
                 RWDD1 
                 rwd domain containing 1 
               
               
                 DOCK9 
                 dedicator of cytokinesis 9 
               
               
                 GDAP1 
                 ganglioside-induced differentiation-associated 
               
               
                   
                 protein 1 
               
               
                 AKAP13 
                 lymphoid blast crisis oncogene 
               
               
                 OSBPL6 
                 osbp-related protein 6 
               
               
                 PDE1A 
                 phosphodiesterase 1a, calmodulin-dependent 
               
               
                 DDX47 
                 dead (asp-glu-ala-asp) box polypeptide 47 
               
               
                 MAP3K15 
                 mitogen-activated protein kinase kinase kinase 
               
               
                   
                 15 
               
               
                 KRIT1 
                 krit1, ankyrin repeat containing 
               
               
                 B3GNT6 
                 udp-glcnac:betagal beta-1,3-n- 
               
               
                   
                 acetylglucosaminyltransferase 6 
               
               
                 MBNL2 
                 muscleblind-like 2 ( drosophila ) 
               
               
                 C13orf23 
                 chromosome 13 open reading frame 23 
               
               
                 SPTAN1 
                 spectrin, alpha, non-erythrocytic 1 (alpha- 
               
               
                   
                 fodrin) 
               
               
                 FMNL2 
                 formin-like 2 
               
               
                 KIF21A 
                 kinesin family member 21a 
               
               
                 FNDC3A 
                 fibronectin type iii domain containing 3a 
               
               
                 ARRDC4 
                 arrestin domain containing 4 
               
               
                 TRIM4 
                 tripartite motif containing 4 
               
               
                 ZCCHC8 
                 zinc finger, cchc domain containing 8 
               
               
                 ANXA7 
                 annexin a7 
               
               
                 TRAM1 
                 translocation associated membrane protein 1 
               
               
                 KIAA1411 
                 kiaa1411 
               
               
                 CBLB 
                 cas-br-m (murine) ecotropic retroviral 
               
               
                   
                 transforming sequence b 
               
               
                 SMARCA1 
                 swi/snf related, matrix associated, actin 
               
               
                   
                 dependent regulator of chromatin, subfamily a, 
               
               
                   
                 member 1 
               
               
                 DNAJC12 
                 dnaj (hsp40) homolog, subfamily c, member 
               
               
                   
                 12 
               
               
                 CAPG 
                 capping protein (actin filament), gelsolin-like 
               
               
                 POPDC3 
                 popeye domain containing 3 
               
               
                 RBM28 
                 rna binding motif protein 28 
               
               
                 GARNL1 
                 kiaa0884 protein 
               
               
                 MARS 
                 methionine-trna synthetase 
               
               
                 SLC43A1 
                 solute carrier family 43, member 1 
               
               
                 SPAG9 
                 sperm associated antigen 9 
               
               
                 BAT2D1 
                 bat2 domain containing 1 
               
               
                 PPHLN1 
                 hypothetical protein hspc206 
               
               
                 PLCL3 
                 phospholipase c-like 3 
               
               
                 IL6R 
                 interleukin 6 receptor 
               
               
                 STC2 
                 stanniocalcin 2 
               
               
                 PSAT1 
                 phosphoserine aminotransferase 1 
               
               
                 COL4A3BP 
                 collagen, type iv, alpha 3 (goodpasture antigen) 
               
               
                   
                 binding protein 
               
               
                 GRHL1 
                 grainyhead-like 1 ( drosophila ) 
               
               
                 RNF41 
                 ring finger protein 41 
               
               
                 GPC2 
                 glypican 2 (cerebroglycan) 
               
               
                 TUBE1 
                 tubulin, epsilon 1 
               
               
                 SEH1L 
                 seh1-like ( s. cerevisiae ) 
               
               
                 BIN1 
                 bridging integrator 1 
               
               
                 NFS1 
                 nfs1 nitrogen fixation 1 ( s. cerevisiae ) 
               
               
                 HRAS 
                 v-ha-ras harvey rat sarcoma viral oncogene 
               
               
                   
                 homolog 
               
               
                 KIAA0350 
                 kiaa0350 protein 
               
               
                 CLIC4 
                 chloride intracellular channel 4 
               
               
                 C6orf107 
                 chromosome 6 open reading frame 107 
               
               
                 PKM2 
                 pyruvate kinase, muscle 
               
               
                 TMEM45A 
                 transmembrane protein 45a 
               
               
                 GARS 
                 glycyl-trna synthetase 
               
               
                 PHF21A 
                 phd finger protein 21a 
               
               
                 CDC42 
                 cell division cycle 42 (gtp binding protein, 
               
               
                   
                 25 kda) 
               
               
                 CEPT1 
                 choline/ethanolamine phosphotransferase 1 
               
               
                 PCK2 
                 phosphoenolpyruvate carboxykinase 2 
               
               
                   
                 (mitochondrial) 
               
               
                 C19orf15 
                 chromosome 19 open reading frame 15 
               
               
                 DNAJA1 
                 dnaj (hsp40) homolog, subfamily a, member 1 
               
               
                 RPS6KA2 
                 ribosomal protein s6 kinase, 90 kda, 
               
               
                   
                 polypeptide 2 
               
               
                 ZNF584 
                 zinc finger protein 584 
               
               
                 FAM102A 
                 family with sequence similarity 102, member a 
               
               
                 H1F0 
                 h1 histone family, member 0 
               
               
                 RBM35A 
                 rna binding motif protein 35a 
               
               
                 BAX 
                 bcl2-associated x protein 
               
               
                 HERPUD1 
                 homocysteine-inducible, endoplasmic 
               
               
                   
                 reticulum stress-inducible, ubiquitin-like 
               
               
                   
                 domain member 1 
               
               
                 AHSA2 
                 aha1, activator of heat shock 90 kda protein 
               
               
                   
                 atpase homolog 2 (yeast) 
               
               
                 TXNL4B 
                 thioredoxin-like 4b 
               
               
                 RTN4 
                 reticulon 4 
               
               
                 BBX 
                 bobby sox homolog ( drosophila ) 
               
               
                 DCC1 
                 defective in sister chromatid cohesion homolog 
               
               
                   
                 1 ( s. cerevisiae ) 
               
               
                 FLJ21908 
                 hypothetical protein flj21908 
               
               
                 TAPBP 
                 tap binding protein (tapasin) 
               
               
                 KIAA1217 
                 kiaa1217 
               
               
                 RIF1 
                 dkfzp434d193 protein 
               
               
                 UBB 
                 ubiquitin b 
               
               
                 ITSN2 
                 sh3 domain protein 1b 
               
               
                 PPIL3 
                 peptidylprolyl isomerase (cyclophilin)-like 3 
               
               
                 BLP1 (TM2D2) 
                 TM2 domain containing 2 
               
               
                 C16orf75 
                 chromosome 16 open reading frame 75 
               
               
                 C4orf32 
                 chromosome 4 open reading frame 32 
               
               
                 C6orf80 
                 chromosome 4 open reading frame 32 
               
               
                 CLPTM1L 
                 CLPTM1-like 
               
               
                 FAM122C 
                 family with sequence similarity 122C 
               
               
                 FAM129A 
                 family with sequence similarity 129, member A 
               
               
                 FLJ20171 (RBM35A) 
                 RNA binding motif protein 35A 
               
               
                 LOC90529 (C1orf201) 
                 chromosome 1 open reading frame 201 
               
               
                 MKX 
                 mohawk homeobox 
               
               
                 NBLA10383 (CDRT4) 
                 CMT1A duplicated region transcript 4 
               
               
                 TBC1D9B 
                 TBC1 domain family, member 9B 
               
               
                 WIPI49 
                 WD repeat domain, phosphoinositide 
               
               
                   
                 interacting 1 
               
               
                   
               
             
          
         
       
     
         [0052]    (2) Reporter 
         [0053]    The PTB target may be operably linked to a nucleotide sequence that encodes a reporter. The reporter may be green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), red fluorescent protein, monomeric red fluorescent protein, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), bluefluorescent protein (BFP), cycle 3 GFP, Emerald GFP, β-galactosidase, luciferase, chloramphenyl acetyltransferase (CAT), GUS (β-glucuronidase), and/or a variant or fragment thereof. 
         [0054]    A first gene, or minigene, target construct may be constructed such that when the gene or minigene is properly spliced, the downstream reporter coding sequence may be in frame. Expression of such a minigene will produce a reporter output signal. However, when a middle exon, exon 10 of a minigene comprising exons 9 through 11 of PTBP2 for example, is spliced out of the transcript, or skipped over, a frameshift may occur such that the downstream reporter coding sequence is out of frame and a reporter output signal will not be produced. 
         [0055]    A second gene, or minigene, construct may be constructed such that the downstream reporter is out of frame when all exons are splice together, but are in frame when a middle exon is skipped from the transcript. 
         [0056]    An exon may be modified. The modified exon may comprise a frameshifting nucleotide. The frameshifting nucleotide may result in a stop codon that is incorporated into the mRNA sequence. The frameshifting nucleotide may result in a stop codon that is incorporated into the mRNA sequence, if the exon is included in the spliced transcript. The frameshifting nucleotide may result in a stop codon that is incorporated into the mRNA sequence, if the exon is excluded from the spliced transcript. 
         [0057]    The gene or minigene may be modified. The reporter may not be expressed if a modified exon is included in the spliced transcript. The modified exon may be upstream of the reporter. The reporter may be expressed, if the modified exon is excluded from the resultant mRNA. Translation of the modified exon comprising a frameshifting nucleotide may result in the reporter not being expressed. 
         [0058]    The gene or minigene may be modified such that the reporter may not be expressed if an exon is excluded from the resultant mRNA. The modified minigene may result in a frameshift in a downstream reading frame when the exon is skipped from the resultant mRNA. The frameshift may result in the presence of a stop codon. The modification may be upstream of the reporter. The stop codon may be upstream of the reporter. 
         [0059]    c. Candidate Modulator Compound 
         [0060]    The method may use a candidate modulator compound. The candidate modulator may be a candidate for modulating PTB. The cell may express the candidate modulator compound, wherein the expressed candidate modulator compound is in contact with the cell. The expressed candidate modulator compound may be expressed and then secreted from the cell. The secreted candidate modulator compound may be in contact with the cell. The method may comprise stimulating the cell to express the candidate modulator compound. The method may cause the cell to take up the candidate modulator compound. 
         [0061]    The candidate modulator compound may be expressed from a vector or plasmid. The candidate modulator compound may be expressed from a vector or plasmid in the cell. Expression may be controlled via a promoter. The promoter may be inducible. The expressed candidate modulator compound may be secreted from the cell. The candidate modulator compound may be a member of a library to be screened using the method herein described. The library may be combinatorial. A candidate modulator compound may be an antibody, a small compound or molecule, a drug, a peptide, a nucleic acid, an oligosaccharide, or an inorganic compound. The nucleic acid may be a siRNA. 
         [0062]    d. Cell 
         [0063]    The cell may be any cell. The cell may be capable of propagating and/or expressing a vector or plasmid. The cell may be eukaryotic or prokaryotic. The eukaryotic or prokaryotic cell may be living. The eukaryotic cell may be mammalian. The mammalian cell may be a HeLa cell, a CHO cell, a human embryonic kidney cell, or a cancer cell. The human embryonic kidney cell may be a HEK 293 cell or a HEK 293T cell. The cancer cell may be an ovarian cancer cell or a breast cancer cell. The mammalian cell may be from, or in, a sample. The sample may be from a subject. 
         [0064]    (1) Sample 
         [0065]    The sample may be a subject sample and/or a control sample. The sample may comprise nucleic acid and/or protein from a subject. The nucleic acid may be DNA or RNA. The nucleic acid may be genomic. The sample may be used directly as obtained from the subject or following pretreatment to modify a character of the sample. Pretreatment may include extraction, concentration, inactivation of interfering components, and/or the addition of reagents. The subject and control sample may be derived from the same organism but may also be derived from different organisms/individuals. The subject sample may comprise tissue cultures or cell cultures. The subject and/or control sample may comprise the same kind of cell(s) and/or tissue(s). 
         [0066]    Any cell type, tissue, or bodily fluid may be utilized to obtain a sample. Such cell types, tissues, and fluid may include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, blood, plasma, serum, sputum, stool, tears, mucus, saliva, hair, and skin. Cell types and tissues may also include gastrointestinal cells or fluid, inflammatory tissue, premalignant adenomas, colorectal cancer, lymph fluid, ascetic fluid, gynecological fluid, urine, peritoneal fluid, cerebrospinal fluid, a fluid collected by vaginal rinsing, or a fluid collected by vaginal flushing. A tissue or cell type may be provided by removing a sample of cells from an organism, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose). Archival tissues, such as those having treatment or outcome history, may also be used. The tissue may be an ovarian cancer tissue, a breast cancer tissue, a prostate cancer tissue, a lung cancer tissue, a gastric cancer tissue, a small intestine cancer tissue, and/or an inflamed tissue. The sample may be frozen, formalin-fixed, and/or paraffin-embedded. Nucleic acid purification may not be necessary. 
         [0067]    (2) Subject 
         [0068]    The subject may be a mammal. The mammal may be a human. The human may be healthy. The human may not exhibit symptoms of an illness. The human may be ill. The illness may be symptomatic of a disease. The illness may be elevated fever, high body temperature, low body temperature, hair loss, hyperpigmentation, skin rash, painful skin rash, fragile thin skin, skin that bruises easily, acne, sun sensitivity, skin thickening, skin ulcers, dry eyes, blurred vision, optic neuritis, eye discomfort or pain, dry mouth, hoarseness, difficulty in swallowing, mouth and/or nose sores, fullness or pressure, choking sensation in throat, chronic fatigue, insomnia, pain or tenderness throughout body, joint stiffness, deformed joints, carpal-tunnel syndrome, Raynaud&#39;s phenomenon (extreme sensitivity to cold in the hands and feet), swelling in hands and feet, weight loss, weight gain, weight gain in upper body or abdomen, rounded or puffy face, lack of coordination or unsteady gait, increased thirst, low blood pressure, high blood pressure, increased urination, nausea, vomiting, diarrhea, cysts on ovaries, irregular menstrual periods, recurrent miscarriage, reduced sex drive, decreased fertility, unexplained anemia, high cholesterol, delayed growth, high blood sugar, low blood sugar, and/or an increase in snoring. 
         [0069]    The subject may be diagnosed with having a disease. The subject may be diagnosed as having a predisposition to develop a disease. The subject may be genetically predisposed to develop a disease. The disease may be cancer. The cancer may be brain, breast, skin, stomach, prostate, lung, and/or ovarian cancer. The ovarian cancer may be epithelial ovarian cancer (EOC). 
         [0070]    (3) Vector 
         [0071]    A vector may be used to express the PTB target gene, PTB target minigene, and/or the candidate modulator compound. The vector may be an expression vector. The expression vector may comprise one or more control sequences capable of enhancing, increasing, attenuating, suppressing, or inhibiting, the expression of the PTB target gene, PTB target minigene, and/or the candidate modulator compound. Control sequences that are suitable for expression in prokaryotes, for example, include a promoter sequence, an operator sequence, and a ribosome binding site. Control sequences for expression in eukaryotic cells may include a promoter, an enhancer, and a transcription termination sequence (i.e. a polyadenylation signal). 
         [0072]    The expression vector may include other sequences. Expression vectors may comprise inducible or cell-type-specific promoters, enhancers or repressors, introns, polyadenylation signals, selectable markers, polylinkers, site-specific recombination sequences, and other features to improve functionality, convenience of use, and control over mRNA and/or protein expression levels. A signal sequence may direct the secretion of a polypeptide fused thereto from a cell expressing the protein. In the expression vector, nucleic acid encoding a signal sequence may be linked to a polypeptide coding sequence so as to preserve the reading frame of the polypeptide coding sequence. 
         [0073]    The vector may be a plasmid, a phage, and/or a virus. The vector may be modified. The vector may be a lentiviral vector. 
         [0074]    e. Control 
         [0075]    The control may be PTB-expression or activity associated in a second cell. The control second cell may comprise PTB and the reporter system. The control second cell may be contacted with a modulator compound known to induce or enhance or suppress or inhibit or inhibit completely PTB-expression or activity. 
         [0076]    The level(s) of expression and/or activity of PTB in a cell in contact with, or formerly in contact with, may be compared to the level(s) of expression and/or activity of PTB in the control second cell. 
         [0077]    The control may be another PTB target gene or minigene. For example, the PTB target gene or minigene may be a second, third, fourth, fifth, or sixth or more target gene. The other PTB target gene or minigene may comprise the PTB target gene operably linked to a reporter sequence. The level(s) of reporter output may be indicative of PTB activity or lack thereof. 
         [0078]    The control may be a cell treated with DMSO, which may serve as a negative control. The control may be a cell that expresses doxycycline-induced PTBsiRNA, which may serve as a positive control. 
         [0079]    f. Recovery of Modulator Compound 
         [0080]    Methods for recovery of the candidate modulator compound identified as modulating PTB expression and/or activity may vary depending on the expression system employed. A compound including a signal sequence may be recovered from the culture medium or the periplasm. The compound may be expressed intracellularly and recovered from the culture medium. 
         [0081]    The expressed modulator compound, or candidate modulator compound, may be purified from culture medium or a cell lysate by any method capable of separating the compound from one or more components of the host cell or culture medium. The compound may be separated from host cell and/or culture medium components that would interfere with the intended use of the compound. As a first step, the culture medium or cell lysate may be centrifuged or filtered to remove cellular debris. The supernatant may then be concentrated or diluted to a desired volume or diafiltered into a suitable buffer to condition the preparation for further purification. The compound may then be further purified. The compound may be purified using an affinity column containing the cognate binding partner of a binding member of the compound. A compound fused with GFP, hemaglutinin, or FLAG epitope tags or with hexahistidine or similar metal affinity tags may be purified by fractionation on an affinity column. 
         [0082]    Compounds identified by the herein described method may be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support. For recovery of an expressed candidate compound, the host cell may be cultured under conditions suitable for cell growth and expression and the expressed compound recovered from a cell lysate or, if the candidate compounds are secreted, from the culture medium. The nutrients and growth factors are, in many cases, well known or may be readily determined empirically by those skilled in the art. Suitable culture conditions for mammalian host cells may be described in “Mammalian Cell Culture” (Mather ed., Plenum Press 1984) and in Barnes and Sato (Cell, 22:649 (1980)). 
       3. METHOD OF TREATING DISEASE 
       [0083]    Also provided herein is a method for treating a subject diagnosed with, or predisposed to, a disease. The method may comprise administering the PTB modulator compound identified by the method described herein to a subject in need thereof. The subject may be a mammal. The mammal may be a human. 
         [0084]    The subject may be diagnosed with having a disease. The subject may be diagnosed as having a predisposition to develop a disease. The subject may be genetically predisposed to develop a disease. The disease may be cancer. The cancer may be brain, breast, skin, stomach, prostate, lung, and/or ovarian cancer. The ovarian cancer may be epithelial ovarian cancer (EOC). 
         [0085]    The present invention has multiple aspects, illustrated by the following non-limiting examples. 
       EXAMPLES 
     Example 1 
     PTB Overexpression in Ovarian Tumors 
       [0086]    Epithelial ovarian tumors overexpress PTB compared to their matched normal ovarian tissues. See PCT/U.S.07/07352, which is herein fully incorporated by reference. Based upon this finding, PTB expression in ovarian tumors with different malignancy and in invasive epithelial ovarian cancer (EOC) at different stages was evaluated. Two specialized tissue microarrays (TMAs), on (called Ovarian Disease Status TMA) containing benign ovarian tumors, borderline/low malignant potential (LMP) ovarian tumors as well as invasive EOC, and the other (called Ovarian Cancer Stage TMA) containing invasive EOC ranging from stage Ito stage IV disease, were evaluated by immunohistochemical staining for PTB. The result of average staining in the Ovarian Disease Status TMA is summarized in Table 3. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Table 1. Average PTB staining in the Ovarian Disease Status TMA 
               
             
          
           
               
                   
                 Overall Expression † ‡ 
               
             
          
           
               
                   
                 All 
                   
                 All 
                   
               
               
                 Disease Status 
                 Negative 
                 Mixed 
                 Positive 
                 Total 
               
               
                   
               
             
          
           
               
                 Benign 
                  8 (47.1) 
                  6 (35.3) 
                  3 (17.6) 
                 17 
               
               
                 Borderline/LMP 
                  8 (16.3) 
                 12 (24.5) 
                 29 (59.2) 
                 49 
               
               
                 Invasive 
                  2 (3.0) 
                  8 (11.9) 
                 57 (85.1) 
                 67 
               
               
                 Total 
                 18 
                 26 
                 89 
                 133 
               
               
                   
               
               
                 
                           
                 
               
               
                             indicates data missing or illegible when filed 
               
             
          
         
       
     
         [0087]    In Table 3, the average staining for each valid case (with a minimum of 2 satisfactory cores) is categorized into three groups: all negative (all evaluable cores were negative), all positive (all evaluable cores were positive), and mixed (at least one evaluable core negative and one evaluable core positive). Row percentage within each staining category is provided within parentheses. Statistical significance was evaluated using Fisher&#39;s exact test (2×2 tables) or its Mehta and Patel version (R×C tables). † Overall test: p=4.2×10-7 for benign vs. borderline/LMP vs. invasive tumors. ‡ Pair-wise comparisons: p=4.7×10-8 for benign vs. invasive, p=0.0069 for benign vs. borderline/LMP and p=0.0039 for borderline/LMP vs. invasive tumors. 
         [0088]    As shown in the table, the percentage of cases stained all positive increases while the percentage of cases stained all negative or mixed decreases in the order of benign tumor, borderline/LMP tumor and invasive EOC. Approximately 85% EOC stained all positive for PTB, whereas a great majority of benign ovarian tumors stained all negative or mixed with only 17.6% stained all positive. The percentages of borderline/LMP ovarian tumors that stained all positive, all negative, or mixed fell between those of benign and invasive tumors. Statistical analyses indicated that the differences in PTB staining amoung benign, borderline?LMP and invasive ovarian tumors were significant in both overall comparison and all pair-wise comparisons. Analysis focusing on one subtype, i.e. mucinous tumors, generated the same results as above. In contrast, staining of Ovarian Cancer Stage TMA showed that great majority of cases were stained all positive and none stained all negative for PTB and there were no significant differences in average staining or frequency of positive cancer cell between any tumor stages. PTB expression may be associated with malignancy of ovarian tumors but not with stage of EOC. 
       Example 2 
     Immortalization of Ovarian Epithelial Cells Increases the Expression of PTB 
       [0089]    Expression of PTB was examined via western blot in normal human ovarian surface epithelia (HOSE), life-extended HOSE (105E398, HOSE transduced by SV40 T-antigen), truly immortalized HOSE (IOSE120T, HOSE sequentially transduced by SV40 T-antigen and hTERT) and ovarian epithelial tumor cell lines PA-1, SKOV3, OVCAR8 and A2780. As shown in  FIG. 1A , the expression of PTB is substantially overexpressed in life-extended IOSE398 cells and maintained at high levels in IOSE120T and ovarian tumor cell lines, compared to normal HOSE cells. Further, we compared the PTB levels at different passages of IOSE398 cells, which senesce at around passage 20. As can be seen in  FIG. 1B , PTB levels were gradually reduced when cells were approaching senescence. The up-regulation of PTB may be an early event in the neoplastic transformation of ovarian epithelial cells and may be required for cell growth.  FIG. 1A  shows immunoblotting analysis of PTB expression in cell lines.  FIG. 1B  shows PTB expression in IOSE398 cells at different passages. Multiple PTB bands are different splice variants of PTB. The expression levels of PTB are quantified as a ratio of PTB to β-actin expression. 
       Example 3 
     Knockdown of PTB Suppresses Ovarian Tumor Cell Growth Both In Vitro and In Vivo 
       [0090]    siRNA technology was .used to knock down the expression of PTB in tumor cells and to examine the effects of such manipulations on cell growth and malignant properties. Three siRNA (PTBsi1, PTBsi2, and PTBsi3) sequences targeting different regions of PTB mRNA were used. A siRNA can be generated as described in PCT/US2007/007352, which is herein fully incorporated by reference. Each of PTBsi1, PTBsi2, and PTBsi3 may be generated in a cell from a shRNA, which is formed after transcription of its coding sequence. The sequences of three pairs of oligonucleotides encoding for PTB shRNA1, shRNA2, and shRNA3 are shown in Table 3. The annealing of two oligonucleotides generates a DNA fragment with protruding ends compatible with Hind III and Bgl II restriction enzyme sites respectively. The coding sequences for each siRNA were cloned individually into a lentiviral vector downstream of H1 promoter and tetO element and thus the expression of these siRNAs is under control of doxycycline (DOX) (i.e. induced by DOX). We established several stable sublines carrying these expression cassettes.  FIG. 2A  shows the knockdown of PTB by DOX-induced siRNAs at both mRNA and protein levels;  FIGS. 2B ,  2 C and  2 D show the suppression of ovarian tumor cell growth, colony formation in soft agar and invasiveness by PTB knockdown. We have also examined whether knockdown of PTB could influence ovarian tumor cell growth in xenograft mouse model. Athymic nu/nu mice were inoculated A2780 sublines expressing DOX-induced PTB siRNA (A2780/PTBsi1 and A2780/PTBsi3) or luciferase siRNA (A2780/LUCsi) subcutaneously and then treated in three different ways: No DOX, with DOX or No DOX for 12 days and then with DOX.  FIG. 3  shows the results of this in vivo experiment. It can be seen that tumors originated from sublines A2780/PTBsi1 and A2780/PTBsi3 grew slower in mice administered DOX either from the beginning of inoculation or 12 days after inoculation. Knockdown of PTB may suppress tumor growth in vivo. With respect to  FIG. 2C , the upper part of the figure shows colonies in soft agar and the lower part shows average ratios (in percentage) of colony numbers formed with DOX vs. without DOX (n=3). With respect to  FIG. 2D , the upper part shows invasive cells under microscope (40×) of one experiment and lower part shows average ratios (in percentage) of invasive cells grown with DOX vs without DOX (n=3). Invasive cells were counted under microscope with high magnification (150×). Arrows indicate the invasive cells. * and ** indicate P&lt;0.05 and P&lt;0.01, respectively, when compared to either control cell line. Error bars represent the standard error (SE). With respect to  FIG. 3 , Athymic nu/nu mice were injected subcutaneously at both flanks 5×10 6  tumor cells in 0.1 ml PBS. Every mouse was inoculated two different subline cells, one at a flank, to maximize the randomness of distribution of sublines in mice. For each subline, there were 30 injections. After inoculation, mice were treated in three different ways: one group was given drinking water supplemented with 5% sucrose, another group was given drinking water supplemented with 5% sucrose and 2 mg/ml DOX, and the third group was first given drinking water supplemented with 5% sucrose for 12 days and then switched to drinking water supplemented with 5% sucrose and 2 mg/ml DOX for the rest of the experiment. Tumor sizes were measured in long and short dimensions every three or four days. Tumor size was calculated with formula axb2/2, where a=long dimension and b=short dimension. Mice were sacrificed on day 21 and day 23 because many tumors reached pre-defined tumor size limit. 
         [0000]    
       
         
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                   
                 shRNA No. and 
                   
               
               
                   
                 Oligonucleotide 
                   
               
               
                   
                 Sequences 
                 SEQ ID NO. 
               
               
                   
               
             
             
               
                   
                 shRNA1: 
                 SEQ ID NO: 1 
               
               
                   
                 5′ GATCCCCAGGTGACAGC 
                   
               
               
                   
                 CGAAGTGCATTCAAGAGA 
                   
               
               
                   
                 TGCACTTCGGCTGTCACCT 
                   
               
               
                   
                 TTTTTGGAAA 3′ 
                   
               
               
                   
                 and 
                   
               
               
                   
               
               
                   
                 5′ AGCTTTTCCAAAAATGCA 
                 SEQ ID NO: 2 
               
               
                   
                 CTTCGGCTGTCACCTTCTCT 
                   
               
               
                   
                 TGAAAGGTGACAGCCGAAG 
                   
               
               
                   
                 TGCAGGG 3′ 
                   
               
               
                   
               
               
                   
                 shRNA2: 
                 SEQ ID NO: 3 
               
               
                   
                 5′ GATCCCCAACTTCCATCAT 
                   
               
               
                   
                 TCCAGAGAATTCAAGAGATT 
                   
               
               
                   
                 CTCTGGAATGATGGAAGTTT 
                   
               
               
                   
                 TTGGAAA 3′ 
                   
               
               
                   
                 and 
                   
               
               
                   
               
               
                   
                 5′ AGCTTTTCCAAAAATTCT 
                 SEQ ID NO: 4 
               
               
                   
                 CTGGAATGATGGAAGTCTC 
                   
               
               
                   
                 TTGAAAACTTCCATCATTCC 
                   
               
               
                   
                 AGAGAAGGG 3′ 
                   
               
               
                   
               
               
                   
                 shRNA3: 
                 SEQ ID NO: 5 
               
               
                   
                 5′ GATCCCTGACAAGAGCCG 
                   
               
               
                   
                 TGACTACTTCAAGAGAGTAG 
                   
               
               
                   
                 TCACGGCTCTTGTCATTTTTG 
                   
               
               
                   
                 GAAA 3′ 
                   
               
               
                   
                 and 
                   
               
               
                   
               
               
                   
                 5′ AGCTTTTCCAAAAATGACAA 
                 SEQ ID NO: 6 
               
               
                   
                 GAGCCGTGACTACTCTCTTGAA 
                   
               
               
                   
                 GTAGTCACGGCTCTTGTCAGGG 3′ 
                   
               
               
                   
               
             
          
         
       
     
       Example 4 
     Knockdown of PTB Alters Splicing Pattern and Expression Profile in the Cell 
       [0091]    Microarray analyses of genome-wide splicing patterns and gene expression profiling were performed in A2780/PTBsi3 cells with or without PTB knockdown. Gene expression profiling was assessed using Affymetrix HG-U133 plus 2 oligonucleotide arrays, and the splicing pattern was assessed by Jivan Biologics&#39; splicing-sensitive microarrays. The former analysis was done three times and the latter was done twice with total RNAs isolated from separate experiments. We also examined the gene expression profile once in the control subline, A2780/LUCsi, grown with or without DOX. 
         [0092]    DOX treatment caused very few changes in gene expression of the controls. With signal intensity of 200 as a cutoff, only 7 genes were found to be changed more than 2-fold, among which 3 have annotation data available and other 4 do not. However, in A2780/PTBsi3 cells treated with DOX (i.e. PTB knockdown), 52 genes were found consistently changed more than 2-fold in all three separate experiments. As an indication of the reliability of the assay, PTB was among these genes and consistently downregulated about 4-fold in all three experiments. Between these genes and those identified in the control cell line, there was one in common, which was dramatically increased in all DOX treated cells, indicating it was not induced by PTB knockdown. In total, we found 50 genes whose expression was regulated by PTB. Among these genes, 27 were downregulated and 23 were upregulated. We have validated these changes in a few of these genes (INHBE, CDC42, PTBP2 and PKP4) by real-time PCR or conventional RT-PCR. As an example,  FIG. 4A  shows the result of validation of INHBE expression. 
         [0093]    From the splicing-sensitive microarray analysis, we identified 317 genes whose splicing patterns were consistently altered more than 2-fold after PTB knockdown in two separate experiments. Table 4 shows the summary of these alterations. The results indicate that PTB&#39;s role in alternative splicing is much broader than previously thought as a splicing repressor enhancing exon skipping. It actually participates in all kinds of splicing events including exon skipping, alternate use of exons, exon trimming and intron retention. It is worth noting that many genes underwent changes in two or more splicing events after PTB knockdown. Therefore, the total number of genes is less than the sum of individual numbers in the Table. Seventeen genes with altered expression levels by Affymetrix microarray analyses were also found altered in their splicing patterns, so it is very likely that the changes in mRNA levels of some genes may reflect switches in splicing patterns rather than transcription. We have picked seven differentially expressed splice variants found by the microarray analysis in order to further examine their expression using conventional RT-PCR and have validated six, as shown in  FIG. 4B . We also validated PICALM at protein level ( FIG. 4C ). These splice variants are all new targets of PTB regulation of alternative splicing not previously reported. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Table 2. Summary of alternative splicing influenced by PTB 
               
             
          
           
               
                   
                   
                 Genes with 2 or more fold change in two 
               
               
                   
                 Splicing events 
                 experiments 
               
               
                   
                   
               
             
          
           
               
                   
                 ExonSkip 
                 190 
               
               
                   
                 Alt1stExon 
                 62 
               
               
                   
                 AltLastExon 
                 48 
               
               
                   
                 Alt3Donor 
                 16 
               
               
                   
                 Alt5Acceptor 
                 14 
               
               
                   
                 IntronRetention 
                 21 
               
               
                   
                 Total unique genes 
                 317 
               
               
                   
                   
               
             
          
         
       
     
       Example 5 
     A Reporter System for Detection of PTB Activity in the Cell 
       [0094]    The results presented above (see  FIGS. 2 and 3 ) suggest that PTB may be a good therapeutic target for ovarian cancer. In this application, we intend to test this idea by high throughput screening for small molecules that inhibit PTB activity in the cell. In order to do this, we have begun to develop a reporter system to monitor the activity of PTB. As described earlier, a major function of PTB is to regulate alternative splicing. Accordingly, our reporter system monitors PTB activity by detecting alternative splicing regulated by PTB. As shown in  FIG. 4B , splicing of PTBP2 exon 10 was controlled by PTB. Knockdown of PTB almost completely suppressed skipping of exon 10, resulting in a single transcript with exon 10 included. Our reporter system makes use of this characteristic of PTB. It is a minigene construct containing the genomic sequence spanning exon 9 to exon 11 of PTBP2, which is immediately upstream of the coding sequence of dsRed or EGFP.  FIG. 5A  shows the schematic diagram of the construct. The genomic sequence was obtained by PCR from A2780 cell genomic DNA with a start codon, ATG, added at 5′ end of the exon 9. Exon 10 is composed of 34 nucleotides and its skipping will cause a shift of the downstream reading frame. We engineered one construct (called PTBP2-dsRed) so that the downstream dsRed coding sequence will be in-frame when all three PTBP2 exons are spliced together and out of frame when exon is 10 skipped. In the other construct (called PTBP2-EGFP), we engineered it so that the downstream EGFP is out of frame when all three exons are spliced together but will be in frame when exon 10 is skipped. Therefore, the former construct can report the existence of the big variant with three exons included and the latter construct can report the existence of the small variant with exon 10 skipped. When these two constructs are introduced into cells expressing high levels of PTB, such as 293T cells and A2780 cells, we will see both dsRed and EGFP expressed because both PTBP2 splice variants are generated in such cells.  FIG. 5B  shows the experimental results of co-transfection of these two constructs into 293T cells. RT-PCR indicated that the PTBP2 exons in the constructs were spliced together to form two distinct variants as expected ( FIG. 5C ). These results confirm the feasibility of using these constructs to detect the alternative splicing of PTBP2 exon 10. These constructs may be used to monitor the levels of PTB in the cell. We expect to see an increase of red fluorescence and a decrease of green fluorescence when PTB is knocked down or its activity is inhibited, because under this condition the expression of the big variant will increase while the expression of the small variant will decrease (see  FIG. 4 ). 
         [0095]    As shown above, we have constructed two plasmids to carry the PTBP2 minigene spanning exon 9 to exon 11 and that the minigene can be correctly processed into two distinct splice variants in the cell to tell the splicing status of exon 10. Because the splicing of the exon 10 is controlled by PTB (see  FIG. 4 ), PTB&#39;s expression or activity may be monitored through the detection of splicing of PTBP2 exon 10. To test the capability of these two constructs in this regard, we will first test whether they can monitor the down-regulation of PTB expression by siRNA. If confirmed, we will then optimize this reporter system for later high throughput screening. With respect to  FIG. 5A , a schematic diagram of the constructs to report the alternative splicing of PTBP2 exon 10 is shown. The rectangular bars represent exons and the lines represent the introns. The numbers above them are the length of corresponding exons and introns.  FIG. 5B  shows co-transfection of 293T cell with the two constructs. Left, red fluorescence; middle, green fluorescence; right, merged image.  FIG. 5C  shows RT-PCR of the SVs of PTBP2-dsRed and PTBP2-EGFP. The forward primer is on exon 9 and the reverse primer is on dsRed or EGFP. M: marker; 1: co-transfection with PTBP2-dsRed and PTBP2-EGFP; 2: transfection with PTBP2-dsRed; 3: transfection with PTBP2-EGFP. 
       Example 6 
     Construction of Lentiviral Vectors to Carry PTBP2-dsRed and PTBP2-EGFP and Preparation of Corresponding Lentiviruses 
       [0096]    The backbone of our current constructs is from plasmid pEGFP-N1 (Clontech, Mountain View, Calif.). We cloned the PTBP2 genomic DNA into this vector at the sites EcoRI and BamHI. Because of limitations with transient plasmid transfection such as low efficiency and short retention period, it is more convenient to use lentiviruses to deliver the minigene constructs into the cell and express them. We obtained several lentiviral vectors as well as packaging plasmids from Dr. Didier Trono (University of Geneva, Switzerland). The specific lentiviral vector that will be used to carry and express PTBP2-dsRed and PTBP2-EGFP is LV-tTR/KRAB. We will replace the tTR/KRAB in this vector with PTBP2-EGFP or PTBP2-dsRed to generate LV-PTBP2-EGFP and LV-PTBP2-dsRed, as shown in  FIG. 6 . Regular cloning techniques will be employed to accomplish this. Once these lentiviral vectors are prepared, we will make lentiviruses in HEK293T cells using the second generation packaging system, which includes pMD2G (expressing vesicular stomatitis virus G envelope protein (VSV-G)) and psPAX2 (expressing the components necessary for packaging). We routinely generate high titers of lentiviruses (&gt;10 7 ) without concentration. 
       Example 7 
     Modification of Lentiviral Vectors Carrying the Expression Cassette for DOX-Inducible PTB siRNA 
       [0097]    The vector-based DOX-inducible PTB siRNA will be used to knockdown PTB expression in the cell to test the above reporter system. However, the lentiviral vectors we made previously to express DOX-inducible PTB siRNA or control siRNA also express EGFP and thus are not suitable for this purpose. Therefore, we will replace the coding sequence for EGFP from these vectors with the puromycin resistant gene by regular cloning techniques. The resulting lentiviral vector is depicted in  FIG. 8 . Cells transduced by this vector only will express short hairpin RNA (shRNA) constitutively. If the cells are also transduced by LV-tTR/KRAB, which harbors the expression cassette of a fusion protein of tet repressor and KRAB, then the expression of shRNA become DOX-inducible. 
       Example 8 
     Establishment of Sublines to Express PTBP2-EGFP and PTBP2-dsRed 
       [0098]    In this application, our focus is on ovarian cancer. Therefore, we will use a panel of ovarian cancer cell lines to test the reporter system. In our previous work, A2780 cells were used to study the effects of PTB knockdown. Hence, we will start with this ovarian cancer cell line. Co-infection of A2780 cells with lentiviruses LV-PTBP2-EGFP and LV-PTBP2-dsRed will be done in 12-well plate. Infected cell will then be seeded into 10 cm-dishes at very low density (300 to 400 cells per dish) to allow the formation of colonies. Cell colonies exhibiting both red and green fluorescence will be picked and expanded. Expression of long and short SVs of PTBP2-dsRed and PTBP2-EGFP in these cell clones will be further confirmed by RT-PCR as we did in  FIG. 5C . Other ovarian cancer cell lines to be tested are OVCAR3, OVCAR4, OVCAR5, OVCAR8, IGR-OV1 and SKOV3. They belong to NCI 60 human cancer cell lines used for in vitro drug screening and are different in their phenotypes such as responses to hormones or chemotherapeutic agents. The ideal cell lines for high throughput screening, to be determined here, will be those expressing low intensity of red fluorescence and high intensity of green fluorescence. We will use optical filter sets with excitation/emission wave lengths (nm) 558/583 and 488/507 to detect dsRed and EGFP fluorescent intensity, respectively, in all experiments in this application. 
         [0099]    There are two ways to knock down PTB expression by siRNA in the cells. One is to express PTB siRNA constitutively and the other is to express DOX-induced PTB siRNA. For the purpose of HTS, it is better to test the reporter system with the second way because it resembles the drug treatment in HTS the best. Therefore, we will establish secondary sublines to express DOX-inducible PTB siRNA on the basis of the above sublines. Cells of the above sublines will be co-infected by lentiviruses carrying PTB siRNA or luciferase (LUC) siRNA (see  FIG. 7 ) and lentiviruses carrying tTR/KRAB, which binds to the tetO to inhibit the expression of downstream gene. Cell colonies formed after puromycin selection will be picked and expanded. Afterwards, these cells will be examined with Western Blot for the downregulation of PTB by DOX induction (2 μg/ml). Clones with greater than 75% PTB knockdown after DOX induction for 5 days will be further examined to determine the time-course and the dose-response relationship of DOX induction. The sublines displaying approximate linear time-course and dose-response on normal or semi-log graph in DOX-induced PTB knockdown in a 7 days&#39; window and dose range of 10-4 to 2 μg/ml will be subject to further test described below. 
       Example 9 
     Reporter System Test 
       [0100]    Once the above secondary sublines are established, we will test how well they can monitor the downregulation of PTB by siRNA. Initially, we will perform this test in 12-well plate. After we identify the best clones, we will test them in 96-well and 384-well plate for HTS (see below). Cells will be seeded into the wells and DOX will be added right after. The intensity of red and green fluorescence will be measured in a fluorescent plate reader at 24, 48, 72, 96, 120, 144 and 168 hours after DOX addition. The expected result is diagramed in  FIG. 8 . In the cells expressing DOX-inducible PTB siRNA, after DOX addition, red fluorescence will be intensified while green fluorescence diminished, because the long form PTBP2-dsRed is to be upregulated and short form PTBP2-EGFP downregulated by PTB knockdown, and thus the ratio of red fluorescence vs green fluorescence will increase. In contrast, in control cells, DOX addition will not change the intensity of red or green fluorescence and thus the ratio is suppose to stay the same. After the measurement of fluorescent intensity, cells will be harvested and PTB protein levels will be analyzed by immunoblotting. The correlations between PTB levels and intensities of red and green fluorescence will then be established. The ideal clones should display negative correlation between PTB levels and red fluorescent intensity (i.e. more PTB, low red fluorescence; less PTB, high red fluorescence) and positive correlation between PTB levels and green fluorescent intensity. Nonetheless, the clones displaying only negative correlation between PTB levels and red fluorescence should be also useful, because the reduction of the PTBP2-EGFP might be slow even though its production is decreased when PTB is knocked down. In this situation, the ratio will still increase. 
       Example 10 
     Adaptation and Optimization of the Reporter System for High Throughput Screening (HTS) 
       [0101]    In order to apply the above reporter system to HTS, it is necessary to adapt it to 96-well or 384-well microtiter plate format so that it can then be automated for large-scale HTS. As described above, the assay with the reporter system consists of basic four steps: 1.) seeding of cells, 2.) addition of DOX (for positive control) or compounds (from libraries) or DMSO alone (for negative control), 3.) incubation of cells and 4.) measurement of fluorescent intensity. Therefore, adaptation will revolve around the optimization of each of these steps. Specifically, we will perform experiments to address issues about solvent tolerance, optimal cell seeding density, plate uniformity and reproducibility. The primary statistical parameters we will use to judge the results and quality of the assay development are signal-to-background ratio and the Z′ factor. We describe these parameters in more detail in the data analysis section below. 
         [0102]    For 96-well plates, we plan to test four seeding cell densities: 250, 500, 1000 and 2000 cells in 100 μl medium per well. For 384-well plates, the seeding cell densities will be 100, 200, 400 and 800 cells in 30 μl per well. Twenty-four h after seeding, DOX in 1×PBS or 1×PBS only will be added to the cultures at minimum concentration that gives rise to the greatest knockdown of PTB (it is determined in D.1.5 above). The fluorescent intensity of dsRed and EGFP will be monitored daily until 7 days after DOX addition. 
         [0103]    Because small molecular compounds in libraries are dissolved in DMSO, adding the compounds directly to the cultures will also introduce DMSO. Therefore, it is necessary to assess the influence of low concentration of DMSO on cell growth and signal detection. Since compound libraries are typically composed of solutions of 10 mM compound dissolved in 100% DMSO, the dilution range of the compound into the cell culture is usually between 200 to 1000 times, the final concentration of DMSO in cell culture is between 0.1% and 0.5%. Therefore, we will test DMSO tolerance at these two concentrations. 
         [0104]    One of the crucial yet often overlooked components of an HTS facility is data analysis. We have invested a significant amount of time and effort in search of low cost, high return solutions for the facility that all users and collaborators can afford and utilize. We have written our own PerlScript routines for integrating our microplate reader output with our compound library data, and have integrated this data with HTS Benchware software (Tripos) for cluster analysis and hit evaluation. For follow-up assays, we are using the CambridgeSoft BioAsssay software suite for rapid 1050 evaluation. 
         [0105]    The first step in assay optimization and HTS data analysis is to determine and monitor the Z- or Z′-factor of the assay being developed or implemented. This simple statistical parameter was introduced by Zhang et al. to access the quality and utility of any HTS assay. The general equation for the Z-factor is Z′=1 (3σ+control+3σ-control)/lμ+control−μ-controll, where σ is the standard deviation and μ is the mean of signals. This coefficient is reflective of both the dynamic range of the assay signals and the data variation associated with the measurements. Z-factors between 0.5-1.0 indicate an excellent assay with 1.0 being designated as a perfect assay. 
         [0106]    For development and transition of the PTB inhibition assay, Z′ factors will be calculated from data obtained above. For each cell seeding density or each DMSO concentration, at least three tests will be performed and in each test, at least one 96-well or one 384-well plate will be used. The layout of DOX treatment and DMSO treatment in a 96-well plate is shown below in Table 6. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 Row 
                 A 
                 B 
                 C 
                 D 
                 E 
                 F 
                 G 
                 H 
                 I 
                 J 
                 K 
                 L 
               
               
                   
               
             
             
               
                 1 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
               
               
                 2 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
               
               
                 3 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
               
               
                 4 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
               
               
                 5 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
               
               
                 6 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
               
               
                 7 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
               
               
                 8 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
                 DOX 
                 DMSO 
               
               
                   
               
             
          
         
       
     
         [0107]    After measuring fluorescent intensities with appropriate filters, the means and standard deviations of the ratios of red fluorescent intensity/green fluorescence intensity in all DOX-treated wells and all DMSO-treated wells of a plate will be calculated, respectively. With these two statistics, Z′ factors can be calculated using above-mentioned formula. 
         [0108]    The Z′-factors will be monitored continuously when transitioning the assay from the bench, using 96-well plates, to the automated Tecan robot platform that will also be performed first in 96-well plates and then transitioned to 384-well plates if possible. Once the assay is optimized for the maximum Z′-factor, the library will be screened in duplicate. Z′-factors are calculated for each plate during the screening process to ascertain whether or not any problems arise over time, e.g. over the course of hours or days. Each 384-well plate will have 320 compounds, 32 positive controls and 32 negative controls (no compound). 
       Example 11 
     A Reporter System for Detection of PTB Activity in the Cell 
       [0109]    In this system, the minigene construct contains the genomic sequence spanning exon 14 to exon 16 of GABBR1 (gamma-aminobutyric acid (GABA) B receptor, 1) gene, which is immediately upstream of coding sequence of dsRed1 or EGFP. As shown in  FIG. 9 , the splicing of exon 15 of GABBR1 is regulated by PTB expression. In cells without PTB knockdown, the major splice variant of GABBR1 has exon 15 skipped while in cells with PTB knockdown, the major splice variant of GABBR1 has exon 15 included.  FIG. 10  shows the schematic diagram of the construct. The genomic sequence spanning exon 14 to exon 16 of GABBR1 was amplified from human genomic DNA with the start codon ATG added to the 5′ end of exon 14. The amplified genomic fragment was then cloned into pDsRed-N1 and pEGFP-N1 vector between EcoRI and BamHI sites with DsRed or EGFP immediately downstream of GABBR1 genomic sequence. Both resultant expression vectors are expected to express long-form (with exon 15 included) and short-form (exon 15 skipped) GABBR1 splice variants (SVs). Without any sequence modification, DsRed and EGFP are out of reading frame in long-form SVs but in frame in short-form SVs. In order to detect the long-form SV of GABBR1, we cut the resultant expression vector derived from pEGFP-N1 with BamHI enzyme and then removed the overhang ends by mung bean nuclease followed by re-ligation of the plasmid. Such manipulation of the plasmid places the EGFP coding sequence in-frame when expressed with the long-form SV. The expression cassettes constructed above are called GABBR1-DsRed and GABBR1-EGFP, respectively. As shown in  FIG. 11 , after co-transfection of 293T cells with these vectors, we observed the expression of DsRed and EGFP in the cells, which indicates short-form and long-form SVs of GABBR1, respectively. Cells with high levels of PTB should exhibit more of the short-splice form of GABBR1 than the long form, as expressed in greater red fluorescence intensity, whereas in cells with PTB knocked down, we expect to see more long splice forms of GABBR1 than the short form, and will be expressed in more green fluorescence intensity.