# Patent

## Title
Transgenic screen and method for screening modulators of brain-derived neurotrophic factor (BDNF) production

## Abstract

A transgenic screen and method for screening biological and chemical test substances or molecules for their ability to influence or modulate the production of BDNF in cells, includes a fusion gene having a zebrafish BDNF gene fragment (promoter) and a fluorescent marker gene inserted downstream of the BDNF gene fragment. When the fusion gene is injected into a zebrafish embryo, the BDNF promoter causes the production of fluorescent protein in various cell types. The embryo is exposed to a test substance for determining the effect thereof on the production of the fluorescent marker protein.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority on prior U.S. Provisional Application Ser. No. 60/334,079, filed Nov. 30, 2001, and which is incorporated herein in its entirety by reference.


REFERENCE TO SEQUENCE LISTING
The present application incorporates by reference a file named: US 1325-02 Second Substitute Heinrich Sequence Listing, including SEQ ID NO: 1 to SEQ ID NO: 8, provided herewith in a computer readable form—on a diskette, created on Sep. 5, 2006 and containing 12,087 bytes. The sequence listing information recorded on the diskette is identical to the written (on paper) sequence listing provided herein.
BACKGROUND OF THE INVENTION
The present invention is generally directed to screening of genes or modulators, and more particularly to a transgenic screen for screening biological and chemical test substances for their ability to influence or modulate the production of BDNF in cells.
Brain-derived neurotrophic factor (BDNF) belongs to a group of nerve growth factors called neurotrophins (NT). The function of NTs includes fostering the growth and survival of neurons during development. In adult brains, NTs have an influence on neuronal excitability and, specifically, BDNF appears to regulate neuronal morphology and synaptogenesis. It is also known to exhibit neuroprotective effects in a range of central nervous system areas (Binder et al. 2001). BDNF has been shown to enhance motor neuron survival in several experimental animal models (Department of Neurology, Baylor College of Medicine 2001). Neurodegenerative diseases, such as Huntington's Disease, Parkinson's Disease and Alzheimer's Disease are expected to show abnormal BDNF expression. Enhancement of BDNF function is thought to be one of the mechanisms by which anti-depressants work (Russo-Neustadt et al. 2001) and, as such, might have a significant effect in treating depression.
It is believed that raising the level of BDNF production in the cells would be an effective method of treating various neurodegenerative disease conditions. The current screens for substances that modulate BDNF production are based on cell culturing. Therefore, the screens measure the level of BDNF that is secreted into the culture media and measure changes to this level caused by modulators. However, the screens do not measure the change that the modulating substances effect at the transcription level, and may therefore not be as specific in identifying the action of a modulator.
Other work has also linked the BDNF gene promoter to a fluorescent reporter gene that allows screening for agents which affect the reporter gene expression by affecting the BDNF promoter. One such method was in vitro, involving the culture of a transgenic cell line.
A second existing method involves transgenic mice expressing BDNF promoters linked to a reporter gene. Once again, these mice are able to give a readout on substances that modulate gene expression by affecting the BDNF promoter. However, the mice need to be sacrificed to measure the effect of the modulator, or at least a cell culture must be taken. In either case, the advantages of multiple series of dynamic screens on the same test stock are lost.
The conventional screens, methods, or in vitro tests measure BDNF production directly and do not identify the specific transcription mechanism by which production is increased. BDNF expression is the result of a complex process, however, with a number of regulatory (“promoter” or “cis-”) genes regulating the transcription of the neurotrophic factor. The present invention allows screening for the expression of specific genetic segments, to allow researchers to identify factors that affect the activity of specific promoter genes.
OBJECTS AND SUMMARY OF THE INVENTION
The principal object of the present invention is to provide an isolated BDNF gene promoter.
An object of the present invention is to provide a nucleic acid construct.
Another object of the present invention is to provide a nucleic acid construct including a BDNF gene promoter and a fluorescent marker tag.
Yet another object of the present invention is to provide a zebrafish gene construct.
Still yet another object of the present invention is to provide a transgenic zebrafish line capable of expressing BDNF gene promoter.
Still yet another object of the present invention is to provide a cloned zebrafish genomic sequence.
Still yet another object of the present invention is to provide a cloned zebrafish genomic sequence which includes the 5′ UT (untranslated) region of zebrafish BDNF cDNA with its associated promoter.
Still yet another object of the present invention is to provide a transgenic screen for in vivo screening of various biological, inorganic, and organic substances for their ability to modulate the production of BDNF at the transcription level of the BDNF gene in a living organism. The screen includes a zebrafish (Danio rerio) BDNF promoter sequence inserted upstream of a fluorescent marker gene so that the BDNF promoter is marked by fluorescence.
An additional object of the present invention is to provide a transgenic screen which can be used to identify gene targets for drugs for neurodegenerative diseases or to identify biological and chemical substances that directly upregulate BDNF promoters and may therefore have a therapeutic effect on neurodegenerative diseases. Since such substances may also have a neuroprotective effect on patients receiving chemotherapy, the indication thereof would be greatly useful and commercially desirable.
Yet an additional object of the present invention is to provide a transgenic screen which could be formatted for a high throughput screen (HTS).
A further object of the present invention is to provide a method of screening various biological and chemical substances or molecules for their capability to regulate BDNF production in a living organism.
Yet a further object of the present invention is to provide a method of screening various biological and chemical substances for regulation of BDNF production, which does not require cell cultures. Therefore, the effect of potential modulators or substances can be tested on multiple cell and tissue types. The BDNF gene transcription can be measured repeatedly, dynamically, serially, and in multiple screens in individual or groups of living embryos and larvae.
In summary, the main object of the present invention is to provide a transgenic screen and method for screening biological and chemical test substances or molecules for their ability to influence or modulate the production of BDNF in cells, which includes a fusion gene having a zebrafish BDNF gene fragment (promoter) and a fluorescent marker gene inserted downstream of the BDNF gene fragment. When the fusion gene is injected into a zebrafish embryo, the BDNF promoter causes the production of fluorescent protein in various cell types. The embryo is exposed to a test substance for determining the effect thereof on the production of the fluorescent marker protein.



BRIEF DESCRIPTION OF THE DRAWINGS
One of the above objects, novel features and advantages of the present invention will become apparent from the following detailed description of the preferred embodiment(s) of the invention, as illustrated in the drawings, in which:
FIG. 1 illustrates organization of the mammalian BDNF gene;
FIGS. 2A-B illustrate zebrafish BDNF gene BamHI and HindIII subclones and sequencing strategy;
FIG. 3 illustrates restriction digest and southern blot hybridization analyses of genomic clones c206 and 241;
FIG. 4 illustrates apaI RFLP in BDNF gene coding exon;
FIGS. 5A-B illustrate transcription factor recognition sites in the 5′ flank/promoter region (SEQ ID NO: 1);
FIG. 6A illustrates an outline of BDNF/EGFP-F MiniExpress reporter construct of the invention;
FIG. 6B (Panel I) (A-E) illustrates BDNF expression in notochord;
FIG. 6C (Panel II) (A-C) illustrates expression of BDNF/EGFP-F (MiniExpress) in blood vessels of 2-days old embryos; and
FIGS. 7A-E illustrate a nucleic acid sequence of a construct made in accordance with the present invention (SEQ ID NO: 2).



DETAILED DESCRIPTION OF THE INVENTION
The present invention illustrates a cloned zebrafish gene construct and a method of using the same in screening various biological and chemical substances or molecules for their capability to modulate the production of BDNF at the transcription level of the BDNF gene in a living organism.
Zebrafish are useful experimental organisms: small, about 3 cm long, the females can lay hundreds of eggs at weekly intervals. Since fertilization is external, the embryos can be manipulated easily as they are transparent, and examination can be made under the microscope (Wixon 2000). Mutagenesis screens are also easily achieved in the zebrafish, and large-scale projects of this nature have led to the production of huge numbers of mutant lines. Such populations can be useful in identifying genes that interact with the BDNF promoter and are consequently additional targets for modulating BDNF transcription (Huynh and Heinrich 2001).
Transgenic fish lines have been principally used within general scientific research in the analysis of promoter activity through reporter gene expression to identify cis-acting regulatory elements—i.e., the controlling effects of a regulatory gene on a structural gene (Dodd et al. 2000).
BDNF is a member of the neurotrophin family that also includes NGF (Levi-Montalcini, R. 1998, Levi-Montalcini, R. et al. 1995), NT-3 (Maisonpierre, P. C. et al. 1990, Maisonpierre, P. C. et al. 1991), NT-4/5 (Ip, N. Y. et al. 1992), NT-6 (Gotz, R. et al. 1994) and NT-7 (Nilsson, A. S. et al. 1998). BDNF is essential for the development and differentiation of specific sets of peripheral and central neuron in mammals (Alderson, R. F. et al. 1990, Hyman, C. et al. 1991, Johnson, J. E. et al. 1986, Sendtner, M. et al. 1992) and birds (Biffo, S. et al. 1994, Davies, A. M. et al. 1986, Frade, J. M. et al. 1997, Herzog, K. H. et al. 1994, Rodriguez-Tebar, A. and Barde, Y. A. 1988, Rodriguez-Tebar, A. et al. 1989). Like mammals and birds, the fishes possess a unique BDNF gene. Neither the structure nor the function of the fish BDNF gene are presently known.
To prepare tools for the molecular and cellular analysis of BDNF gene structure and function in the fish we used a recently cloned a zebrafish cDNA (Hashimoto, M. and Heinrich, G. 1997). Using the cDNA as a probe, we examined expression of BDNF mRNA in the developing zebrafish embryo and 4 day old larva. We extended this analysis to the earliest stages of embryonic development (Lum and Heinrich, 2001). These analyses showed that, in contrast to mammals, in the zebrafish, BDNF and BDNF mRNA are present in the zygote, and thus, may have a role in stages of development that precede nervous system formation. In the four day old larva BDNF and BDNF mRNA are expressed in specific cells within muscle, heart, neuromast, ear, brain, and cartilage.
Here we report on the cloning and structural analysis of the zebrafish BDNF gene. We show that its intron/exon organization is similar to that of the mammalian BDNF gene. Our genomic clones include the 5′ untranslated region of the previously cloned BDNF cDNA and its associated promoter. When linked to an enhanced green fluorescent protein (EGFP-F) reporter and injected into Zebrafish embryos, this promoter mediates expression in cell types that express the endogenous BDNF gene. Transgenic lines derived from these embryos will allow us to utilize mutagenesis to identify genes that regulate BDNF gene expression.
Materials and Methods
Genomic Library Screening
A genomic PAC (P1 Artificial Chromosome) library was screened by colony hybridization. The library had been constructed from erythrocyte genomic DNA by C. T. Amemiya. The genomic DNA was partially digested with Mbol and ligated into the pCYPAC6 (PAC) vector. Colonies were microarrayed onto nylon filters by the Resource Center of the German Genome Project (Vente, A. et al. 1999, Zehetner, G. and Lehrach, H. 1994). Each 9+9 inch filter contains approximately 24,000 clones (12,000 uniques plus 1 set of duplicates). 4 filters were provided by RZPD GmbH and screened with a mixture of two digoxigenin-labeled BDNF probes. The probes were prepared and labeled by PCR in the presence of digoxigenin-11-dUTP using a previously cloned zBDNF cDNA as template. Probe1 was directed exclusively to the coding exon (exon 2, FIG. 1) and probe2 mainly to the 5′ untranslated region which has sequence similarity to exon 1c of the rat BDNF gene (FIG. 1). The following oligonucleotides were used for PCR. Probe1: sense 5′-acaggttagaagagtgat-3′ (SEQ ID NO: 3) and antisense 5′-cttaatggtcaatgtgca-3′ (SEQ ID NO: 4). Probe2: sense:5′-gctcagtcatgggagtcc-3′ (SEQ ID NO: 5) and antisense 5′-atgaacgaacaggatggtcat-3′ (SEQ ID NO: 6).
FIG. 1 illustrates organization of the mammalian BDNF gene. Boxes designate exons, and lines introns and flanking regions. Open boxes represent 5′ untranslated regions. The arrows indicate the positions of the four promoters which are labeled P1-4. The first 4 exons are alternatively spliced to exon 2 such that BDNF mRNA always includes two exons and always contains exon 2. The first four exons are accordingly labeled 1a-d. The striped box is the translated region of exon 2 that encodes the entire BDNF precursor. The scaly box represents the 3′ untranslated region.
Mapping and Sequencing of Genomic Subclones
Standard restriction enzyme and Southern blot analyses were applied. Sequencing was contracted out to Davis Sequencing. DNA sequencing at the company is performed using ABI Prism 377 DNA sequencers with the 96-lane upgrade. Southern blots were probed with digoxigenin-labeled probes 1 and 2. A third probe (probe3) was used to identify the BamHI subclone encoding the 5′ end of exon 1c (FIG. 2A). FIG. 2B shows the subclones (c2 and c41) that contain the coding exon 2. The following oligonucleotides were used to prepare probe3: sense 5′-ctcaatgcgcactac-3′ (SEQ ID NO: 7) and antisense 5′-ggatcctttggagttgag-3′ (SEQ ID NO: 8).
FIGS. 2A-B illustrate zebrafish BDNF gene BamHI and HindIII subclones and sequencing strategy. Vertical arrows mark restriction sites. Horizontal arrows indicate the origin, length and direction of sequencing reactions.
FIG. 2A shows the subclones that contain exon 1c (c4, c25, and MiBa). It is noted that exon 1c has a BamHI site. The promoter and 5′ flank are located in c25. The 3′ end of exon 1c and the 5′ end of intron 1c are in c4. The sequencing gaps in MiBa are indicated by pairs of slashes. The empty box represents exon 1c. The vertical block arrows point to exon 1c.
FIG. 2B shows the subclones (c2 and c41) that contain the coding exon 2. The black box designates exon 2.
Construction of Fusion Genes
Starting materials were the genomic subclones and a plasmid carrying EGFP-F (pEGFP-F from Clontech, Palo Alto, Calif.). EGFP-F is a derivative of GFP (green fluorescent protein) with enhanced fluorescence and a farnesylation signal at the COOH-terminal derived from the src protein, which anchors EGFP in the cell membrane. Standard methods of restriction enzyme digestion and ligation of selected fragments were applied. Junctions of heterologous fragments were sequenced to confirm correct construction of fusion genes.
Preparation of DNA and Microinjection of Embryos
Plasmids were digested with HindIII and StuI or MluI. The desired restriction fragments were purified by agarose gel electrophoresis. DNA was dissolved in 100 mM KCl at a concentration of 10-50 μg/ml. Phenol red was added to visualize injected DNA solution. DNA was injected into a blastomere or into the cytoplasmic stream below the blastomere(s) at the 2-8 cell stages. Embryos were enzymatically dechorionated with Pronase and extensively washed in embryo medium prior to injection Embryo medium=13 mM NaCl; 4.2 mM NaHCO3; 0.54 mM KCl; 0.025 mM Na2HPO4; 0.044 mM KH2PO4; 1.3 mM CaCl2; 1 mM MgSO4). Embryos were injected and maintained for 14 hpf on a bed of 0.7% agarose in embryo medium. Subsequently, they were placed into deionized water in clear plastic dishes for observation with a Zeiss IM fluorescent microscope or further growth.
Immunocytochemistry
Four (4) days old larvae were fixed in 4% paraformaldehyde. Whole mount embryos were stained with monoclonal Ab C-9 (Santa Cruz Biotech., Santa Cruz, Calif.), raised against a synthetic peptide representing the N-terminal 27 amino acids of human BDNF. Specifically bound Ab was visualized using the ABC system.
In Situ Hybridization
Twenty-four (24) hrs old larvae were fixed in 4% paraformaldehyde. Whole mount embryos were hybridized with a digoxigenin-labeled PCR-generated probe targeted to exon 2 (probe1). Specifically bound probe was visualized with anti-digoxigenin-Fab conjugated to alkaline phosphatase.
Results
Screening of PAC Library
Four (4) 4 filters obtained from the RZPD GmbH (www.rzpd.de) were screened. Each filter contained 12 000 unique clones and 12 000 duplicates. A mixture of two digoxigenin-labeled probes was used. Probe 1 was directed toward the untranslated region of the previously cloned BDNF cDNA, and probe 2 to the coding exon. Two of the 48, 000 clones screened hybridized to the mixture. They were named c206 and c241.
Restriction Enzyme and Southern Blot Hybridization Analysis
FIG. 3 illustrates restriction digest and southern blot hybridization analyses of genomic clones c206 and 241. Plasmid DNA was cut with various restriction enzymes and the digests were subjected to agarose gel electrophoresis in the presence of ethidium bromide. The gel was photographed and the DNA transferred to a nylon membrane. The membrane was probed sequentially with probe1 and probe2. Hybridized probe was visualized with an anti-digoxigenin FAb fragment conjugated to alkaline phosphatase and a chemiluminescent substrate. Panel A is underexposed to show the size marker (HindIII digest of Phage-lambda (lanes 7). Panel C is overexposed to show the smaller restriction fragments. Panel B shows the chemiluminogram. In all panels lanes 1-3 are c206 and lanes 4-6 c241. The DNA was digested with BamHI (lanes 1), HindIII (lanes 2) and EcoRI (lanes 3). The heavy bands in panel B represent hybridization with probe2. The light bands, marked with arrows, represent hybridization to probe1. It is noted that probe2 extends a short distance into exon 2 and therefore weakly hybridizes with DNA fragments containing exon 2.
The corresponding bacterial cultures were also obtained from the RZPD GmbH. Plasmid DNA was extracted from mini-cultures and analyzed by restriction enzyme digestion and southern blotting. The insert lengths were estimated from these digests as 100 kb for c206 and 80 kb for c241. FIG. 3 shows that there are restriction fragments that occur in both clones as well as those that are unique. The unique fragments add up to approximately 10 kb in c206 and 25kb for c241. The overlap is therefore approximately 65 ̃70 kb.
Probe1 and probe2 hybridized to fragments that are common to both genomic clones supporting the conclusion that the genomic clones are authentic. On the other hand, probe1 and probe2 hybridized to different fragments in all three restriction enzyme digests in both clones, suggesting the 5′ UT and coding exons are separated by a considerable distance. It is noted that probe2 overlaps to a small extent with exon 2 and, therefore, hybridizes weakly to the fragments recognized by probe1.
These analyses show that each of the two independent clones contains a complete transcription unit. Since the overlap is about 65 kb the transcription must be 65 kb or less. The human BDNF gene was found to be co-localized with 3 other genes on a 120 kb DNA fragment on chromosome 11p14 (Guillemot, F. et al. 1999).
Subcloning and Sequencing
The genomic clones c206 and c241 were digested with HindIII or BamHI and the mixture of fragments subcloned into the HindIII or BamHI sites of pEGFP-1 (Clontech). Non-fluorescent colonies were collected and screened by dot blot hybridization for subclones hybridizing to probe1, probe2, or probe3. One HindIII subclone hybridized to both probe2 and probe3. This clone was called MiBa. Another HindIII subclone hybridized to probe1, and was called c41. Three BamHI subclones hybridized to probe1, 2 or 3. They were called c2, c25, and c4, respectively. These clones were partially sequenced. (See FIGS. 2A-B for the sequencing strategies and portions that were sequenced.) The nucleotide sequences confirmed that we had cloned the zebrafish BDNF gene.
MiBa contains a 9 kb insert. c25 is completely embedded in MiBa. c4 overlaps with the 3′ end of MiBa and contains an adjacent downstream 1.4 kb HindIII fragment. c41 and c2 contain 3 kb inserts and extensively overlap such that c2 extends only 250 bps farther 3′ than c41. These mapping data are summarized in FIGS. 2A-B.
We have not yet mapped the relative positions of the HindIII or BamHI fragments that contain exon 1c and exon 2. Therefore, the size of intron 1c is not yet known.
RFLP
FIG. 4 illustrates apaI RFLP in BDNF gene coding exon. Genomic subclone c41 and zBDNF cDNA clone 18.1 were digested with restriction enzymes and the fragments separated by agarose gel electrophoresis in the presence of ethidium bromide. The gel was photographed. Lanes 1-2: cDNA clone 18.1 cut with EcoRI (lane 1) and EcoRI/ApaI (lane 2). Lanes 3 and 4: genomic subclone c41 cut with HindIII (lane 3) and HindIII/ApaI (lane 4). Lane 5: Phage lambda HindIII digest.
The nucleotide sequence of c41 revealed an ApaI site in the prepro-region of the BDNF precursor that was not present in the previously cloned cDNA. The presence or absence of the ApaI site was confirmed by restriction analysis. c41 DNA, which contains the coding exon, and BDNF cDNA were cut with a single enzyme (HindIII and EcoRI, resp.) to release the cloned DNA. Aliquots of the digests were then cut with ApaI. The fragments were separated by agarose gel electrophoresis. FIG. 4 shows that the cDNA has no ApaI site and the genomic subclone c41 has a single one. As a result of the single nucleotide difference that abolishes the ApaI site the cDNA encodes a glutamic acid residue just downstream from the signal sequence of the BDNF precursor whereas the genomic clone encodes a Gln residue in the same position. The amino acid substitution alters the negative charge of the side-chain only two residues downstream from an Arg and therefore could be functionally significant. In any event, this RFLP will be useful in future mutagenesis screens when intragenic mutations must be distinguished from extragenic mutations by linkage analysis.
Alternate Promoters
The rat BDNF gene has four independently regulated promoters (see FIG. 1) (Bishop, J. F. et al. 1994, Hayes, V. Y. et al. 1997, Marmigere, F. et al. 1998, Metsis, M. et al. 1993, Nanda, S. and Mack, K. J. 1998, Shintani, A. et al. 1992, Timmusk, T. et al. 1994a, Timmusk, T. et al. 1993, Timmusk, T. et al. 1994b). The associated exons encode four 5′ untranslated tracts which are alternately spliced to the coding exon to generate the mature BDNF mRNA transcripts, all of which encode the identical BDNF precursor. The alternate exons have been designated 1a-c and the coding exon has been exon 2 herein.
Nucleotide sequence comparison of the 5′UT of the previously cloned zebrafish BDNF cDNA with the rat BDNF gene revealed 67% identity with rat exon 1c (Hashimoto, M. and Heinrich, G. 1997). Moreover, in the zBDNF cDNA there was a sudden increase in similarity with the rat gene in the coding region at a point where the rat gene has an intron. This suggested that the zebrafish gene has an intron at the identical position. Sequence analysis of c41, which spans this region, confirmed the presence of an intron precisely where it occurs in the rat. Thus, the exon/intron structure of the BDNF gene is conserved.
In our in situ hybridization analyses of BDNF mRNA expression in 4 days old larvae, we had used probe1 and probe2 (Lum and Heinrich, 2001). The results showed a disparity between cells that hybridized to probe1 that is targeted exclusively to the coding exon (which is common to all BDNF transcripts) and probe2 that is targeted mainly to exon 1c. This disparity suggested that the zBDNF gene, like its rat counterpart, has multiple promoters. If their number and relative arrangement in the transcription unit were conserved we would expect an exon 1d about 500 bps downstream from exon 1c and a pair of exons 1a and b, also about 500 bps apart, located several kb upstream from exon 1c. Since a search through the nucleotide sequence bank at NCBI (National Center for Biological Information, Bethesda, Md.) for sequences similar to zebrafish exon 1c had identified rat exon 1c, we carried out similar searches using all sequences we had obtained from subclones MiBa, c4, and c25. However, none of the searches found any sequences that were related to the rat BDNF gene.
Promoter 3 Analysis
FIGS. 5A-B illustrate transcription factor recognition sites in the 5′ flank/promoter region. The nucleotide sequence was searched for similarities with known transcription factor binding sites using TESS. The nucleotide sequences with similarity were boldfaced and the abbreviated transcription factor name was written above them. When there was overlap between sequences arrows below the sequences were used and the transcription factor names were written either to the left or right of the arrows. The cloned BDNF mRNA sequences are boldfaced, italicized and underlined. The following nucleotide designations are used in TESS: (AC) M; (AG) R; (AT) W; (CG) S; (CT) Y; (GT) K; (AGC) V; (ACT) H; (AGT) H; (CGT) D; (AGTC) X/N. For details on each of the transcription factors, TESS may be consulted.
c25 contains the 5′ end of the previously cloned cDNA and therefore the associated promoter and 5′ flank. To more precisely delineate the promoter region and to facilitate future functional analyses the entire clone was sequenced. The nucleotide sequence was scanned by computer for potential transcription factor binding sites using TESS (‘TESS: Transcription Element Search Software on the WWW’, Jonathan Schug and G. Christian Overton, Technical Report CBIL-TR-1997-1001-v0.0, of the Computational Biology and Informatics Laboratory, School of Medicine, University of Pennsylvania, 1997. As expected, a number of potential binding sites were found. The most relevant sites are shown in FIGS. 5A-B. The mammalian BDNF gene is known to be regulated by calcium (Bishop, J. F. et al. 1997, Finkbeiner, S. 2000, Sano, K. et al. 1996, Shieh, P. B. and Ghosh, A. 1999, Shieh, P. B. et al. 1998) and CREB (Tao, X. et al. 1998). Several AP-1 and a potential CREB recognition sequence were found close to the promoter suggesting the zebrafish may be regulated by similar upstream regulators.
Expression of Promoter 3 in Embryos
FIGS. 6A-C illustrate construction and expression of BDNF and BDNF/EGFP-F (MiniExpress) Fusion genes in zebrafish embryos and larvae.
FIG. 6A illustrates an outline of BDNF/EGFP-F MiniExpress reporter construct of the invention. The 5′ end of exon 1c, consisting of 5′ UT, is fused to the coding sequence of EGFP-F (black box). The 5′ flank extends 1.7 kb upstream. The 3′ end contains the SV40 polyadenylation upstream from the MluI site and SV40 enhancers between the MluI and StuI sites. Arrows indicate sequenced segments.
FIG. 6B. (Panel I) (A-E) illustrates BDNF expression in notochord. Panel IA illustrates BDNF mRNA visualized by in situ hybridization. The probe was a PCR-generated digoxigenin-labeled fragment of exon 2. Panel IB illustrates 4-days old larva. BDNF visualized by immunocytochemistry using MAb C-9 (Santa Cruz Biotech.). Panel IC1 illustrates 2-days old embryo injected with BDNF/EGFP-F (MiniExpress). Superimposed visible and fluorescent images. Panel IC2 illustrates fluorescent image of embryo shown in panel IC1. Panel ID illustrates 2-days old embryo injected with BDNF/EGFP-F (MiniExpress), fluorescent and visible images superimposed. Panel IE illustrates 2-days old embryo injected with BDNF/EGFP-F (MiniExpress), fluorescent image.
FIG. 6C (Panel II) (A-C) illustrates expression of BDNF/EGFP-F (MiniExpress) in blood vessels of 2-days old embryos. Panel IID illustrates expression of BDNF/EGFP-F (MiniExpress) in myocyte of trunk lateral myotome of 2-days old embryo. Panel III (A-C) illustrates expression of BDNF/EGFP-F (MiniExpress) in epithelial cells of 2-days old embryos. Panel IIIA1 is a lateral view, and panel IIIA2 a dorsal view of the same embryo. NC=notochord. BV=blood vessel.
To begin a functional analysis of promoter 1c, the insert of c25 was linked to the EGFP-F reporter. This reporter encodes an EGFP with a farnesylation signal at the 5′ end. As a result, the EGFP becomes membrane anchored. In addition, the EGFP-F sequence is followed by SV40 polyA signals, mRNA 3′ end and enhancers. We chose this reporter because it promised to be significantly more sensitive than EGFP. The resulting construct is shown in FIG. 6A and was called MiniExpress. Vector sequences and various SV40 sequences were removed prior to injection by digestion with StuI or MluI and agarose gel isolation of the desired fragments.
Embryos were dechorionated to facilitate injection of DNA into or close to the blastomeres at the 1-8 blastomere stages. However, even with injections at these early stages expression was highly mosaic, i.e. for any given cell type only a few cells expressed the reporter in any given injected embryo. For this reason, results are only reported for cell types that were seen to consistently express in >10 expressing embryos.
From the above, it can be observed that we have cloned a zebrafish genomic fragment that carries the BDNF coding exon and at least one functional promoter (FIG. 6A). Sequence analysis showed that the intron/exon organization of the zebrafish BDNF gene is identical to that of the mammalian BDNF gene. However, at this time we have identified with certainty only one promoter. Our in situ hybridization analyses with exon-specific probes showed that the exon 1c-specific probe hybridized to only a subset of BDNF mRNA positive cells (Lum and Heinrich, 2001). We also found several size classes of BDNF mRNA on Northern blot hybridization (Hashimoto, M. and Heinrich, G. 1997).
These findings suggest that the zebrafish BDNF gene has multiple promoters. Consistent with this possibility is the fact that promoter 1c expresses in only a subset of BDNF gene expressing cells. On the other hand, it is possible that the construct we examined lacks the cis regulatory elements that are required for expression in the additional cell types that express the endogenous BDNF gene. Again, this possibility are the results of preliminary experiments with constructs that extend farther upstream. Paradoxically, these constructs are expressed in fewer rather than more cell types than MiniExpress. The sequence analyses of MiBa and c4 are 90% complete. The remaining 10% could not be sequenced with the automated methods because they consist of small repetitive sequences and runs of adenosine and thymidine residues. They are thus unlikely to contain expressed exons. The sequenced regions of MiBa and c4 that contain potentially expressed exons together cover about 9 kb of genomic DNA. A BLAST search through the nucleotide sequence banks at NCBI for regions of similarity with the rat BDNF gene failed to find any. It is not clear whether any exons present in the sequenced regions are so dissimilar to their rat counterparts that they cannot be detected by the BLAST search engine, whether they are located farther away, or whether the zebrafish BDNF gene simply does not possess multiple promoters. The question of multiple promoters can be addressed experimentally by rapid amplification of cDNA ends (RACE).
The strong expression of BDNF mRNA in cartilage we had observed in our previous in situ hybridization and immunocytochemical analyses was originally somewhat unexpected (Lum and Heinrich, 2001). The expression of MiniExpress in the notochord that we observed in transiently transgenic embryos here are consistent with these data. The early and strong expression of the BDNF gene suggests an important function in skeleton development. The mammalian BDNF gene is also strongly expressed in cartilage and bone, but its function in these tissues is unknown.
The mammalian BDNF gene utilizes two polyadenylation signals that are almost 4 kb apart (Timmusk, T. et al. 1994, Timmusk, T. et al. 1993a). As a result most BDNF expressing tissues contain a large 4 kb transcript. Timmusk et al. (Timmusk, T. et al. 1994b) showed that this transcript is relatively rare in polysomes compared with the shorter more abundant transcripts of 1.6 kb and thus appears not to be as efficiently translated. We have not observed an equivalent large BDNF mRNA on our Northern blots despite overexposure of the autoradiograms (Hashimoto, M. and Heinrich, G. 1997). The zebrafish BDNF gene, thus appears to utilize only a single polyadenylation signal which we have cloned and sequenced. The 3′ UT of the more abundant 1.6 kb mammalian BDNF mRNA and of zebrafish BDNF mRNA are relatively short, consisting of <500 nucleotides. Interestingly, our BLAST searches found two segments, a 23 and a 42 nucleotide segment, in the 3′UT that are identical in mammalian and zebrafish BDNF messages, suggesting important functions. Indeed, Timmusk et al. (Timmusk, T. et al. 1995) reported that the cloned rat BDNF gene was only expressed cell-specifically in transgenic mice if the 3′ flank was included. However, the fragment they used extended 4 kb downstream from the first polyadenylation signal to the second polyadenylation signal. Therefore, it is not clear whether it is the conserved sequences in the 3′UT of the shorter message that are responsible for the observed cell-specific expression or sequences located yet farther downstream, or both.
We used a reporter derivative of the enhanced green fluorescent protein, EGFP (Harvey, K. J. et al. 2001). A farnesylation signal at the COOH end of this modified EGFP anchors the reporter in the cell membrane. We found this reporter significantly more sensitive that the non-membrane bound EGFP. The membrane anchored EGFP outlines the entire cell membrane. As a result, the identification of the cell type expressing the reporter is greatly facilitated. It was readily possible to distinguish various types of neurons because cell bodies, dendrites and axons were all completely labeled and outlined. For example, primary motor neurons were immediately distinguishable from the primary sensory Rohon-Beard cells (Inoue, A. et al. 1994, Martin, S. C. et al. 1998).
The construct of the invention can be used to rapidly screen a number of substances for their ability to influence the production of BDNF in a living organism. The preferable living organism is a zebrafish embryo or fry. The zebrafish is altered genetically so it carries the new gene that it passes on to all its progeny. The new gene or construct is assembled by standard molecular biology methods. It has two main components: a portion of the zebrafish BDNF gene that controls transcription, i.e., the promoter, and another gene that encodes a protein which fluoresces under UV light. The single new gene derived from the two components is called a fusion gene or construct.
When the fusion gene is injected into a zebrafish embryo, the BDNF promoter portion causes the production of the fluorescent protein in various cell types. The amount of protein, and hence fluorescence, is dependent on the activity of BDNF promoter. One can then expose embryos or larvae that carry this fusion gene to any desired chemical or biological substance to measure the effect of the substance on the production of the FP. The observed fluorescence is a measure of activity of the zebrafish's own BDNF gene and, hence a measure of BDNF production in various organs of the zebrafish. Bu utilizing this kind of screen, one can discover substances that have the capability to modulate BDNF production.
FIGS. 7A-E illustrate a construct made in accordance with the present invention, wherein nucleotides 1 to 263, 2154 to 2172, and 4159 to 6428 represent vectors; nucleotides 2173 to 2967 represent a reporter; nucleotides 264 to 2035 represent 5′ flank of zebrafish BDNF gene; nucleotides 2036 to 2063 represent a promoter of zebrafish BDNF gene; nucleotides 2064 to 2153 exon 1c (5′ UT) of zebrafish BDNF gene; nucleotides 3001-4159 represent SV40 sequences. The fragment injected into zebrafish embryos for expression is represented by nucleotides 236 to 3223.
OPERATION
The main tool are transgenic fish lines that stably express BDNF gene promoters linked to a fluorescent protein reporter (BDNF/FP fusion genes) whose cellular levels can be measured using fluorescent imaging equipment.
In order to create transgenic zebrafish lines, the BDNF/FP fusion genes are constructed from cloned zebrafish BDNF gene promoters and various fluorescent protein (FP) (green, red, yellow or blue) reporters by standard methods. (The FPs are obtained from commercial sources, such as Clontech, Inc.) The fusion genes are sequenced to confirm their structure, and are then injected into zebrafish embryos at the 1-8 cell stage of embryonic development. Transgenic lines are derived from the founder embryos by standard breeding and analysis methods.
Embryos from transgenic lines are exposed to a test substance and the level of reporter FP is compared to controls using fluorescent imaging equipment and computer image analysis. The test substances are either dissolved in ambient water or injected into the yolk or cell mass. At larval stages, the test substances are dissolved in ambient water or injected into various body sites, such as organs, the stomach, or the blood stream. We observed expression in notochord, muscle, epithelial and endothelial cells of the 1 day old embryo in consonance with the endogenous gene.
The construct of the invention and additional constructs, in progress, will allow us to establish transgenic zebrafish lines that will permit direct and live visual observation of BDNF gene expression. Such lines will be useful for the identification of genes that regulate BDNF gene expression using mutagenesis. With a short-lived reporter, it will also be possible to observe “real-time” dynamic changes of BDNF gene transcription in response to various physiological and experimental stimuli in the nervous system of the developing zebrafish embryo.
While this invention has been described as having preferred sequences, ranges, steps, materials, or designs, it is understood that it includes further modifications, variations, uses and/or adaptations thereof following in general the principle of the invention, and including such departures from the present disclosure as those come within the known or customary practice in the art to which the invention pertains, and as may be applied to the central features hereinbeforesetforth, and fall within the scope of the invention and of the limits of the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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## Claims

1. A fusion gene, comprising:
the nucleotide sequence comprising nucleotides 236 to 3223 set forth in SEQ ID NO.: 2.

2. A plasmid comprising the fusion gene of claim 1.

3. An in vitro cell comprising the fusion gene of claim 1.

4. A non-naturally occurring nucleic acid construct, comprising:
the nucleotide sequence as set forth in SEQ ID NO.: 2.

5. A plasmid comprising the construct of claim 4.

6. An in vitro cell comprising the construct of claim 4.