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.

Description of the Invention

REFERENCE TO SEQUENCE LISTING

The present application incorporates by reference a file named: US 1353-03 Heinrich Sequence Listing, including SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4 and SEQ ID NO.: 5, provided herewith in a computer readable form--on a diskette, created on Dec. 22, 2003 and containing 32,891 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.

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.
 

Claim 1 of 5 Claims

1. A transgenic zebrafish embryo or larva, whose genome comprises: a promoter comprising nucleotides 1777 to 1804 as set forth in SEQ ID NO: 4 operably linked to a reporter gene encoding red fluorescence protein (RFP).
 

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