Motoneuronotrophic factor gene sequences

A technology of promoter sequence and nucleic acid sequence, applied in nerve growth factor, growth factor/inducible factor, genetic engineering, etc., can solve the problem of unpublished full-length cDNA, etc.

Inactive Publication Date: 2006-12-13
GENERVON BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] Although multiple biological aspects of MNTF1 have been determined and cDNA fragments encoding pe

Method used

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  • Motoneuronotrophic factor gene sequences
  • Motoneuronotrophic factor gene sequences
  • Motoneuronotrophic factor gene sequences

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] To identify which tissues had the highest MNTF expression, PCR products were amplified from cDNA obtained from a variety of different tissue sources. Adult and fetal cDNA assay sets derived from various tissues were ordered by Clontech, while gene-specific primers were synthesized based on chromosome mapping experiments (see below).

[0075] The following primer sets were used:

[0076] Forward: 5' TTT CTT CCT CCC TAC ATC TCT C 3' (SEQ ID NO: 3)

[0077] Reverse: 5'GAG GGT AAT ATC TGT TGG ATC 3' (SEQ ID NO: 4)

[0078] The expected length of the PCR product was 541 base pairs.

[0079] A.PCR protocol

[0080] 1.Master mix PCR 1X

[0081] 35 μl water

[0082] 5 μl 10X buffer

[0083] 1.5 μl MgCl 2 (50mmol)

[0084] 1 μl dNTPs

[0085] 1 μl of each primer

[0086] 2U Taq Platinum Polymerase

[0087] 5 μl DNA template

[0088] 2. PCR cycle

[0089] 5'95°C

[0090] 38 loops:

[0091] 30"95℃

[0092] 30" annealed at 53°C

[0093] 45" 72°C Extension

[0094] 5...

Embodiment 2

[0100] Total RNA was extracted from the following tissues by the RISO RNA isolation method (Cat. No. 06-200) (Genemed Biotechnologies, Inc., South San Francisco, CA): hippocampus, placenta, brain (fetal and adult brain), retina, pituitary, heart, fetal muscle. This approach combines guanidinium thiocyanate (GTC), a potent and known inhibitor of RNase, with β-mercaptoethanol to slow the rate of RNA degradation. It also disrupts nucleoprotein complexes, allowing RNA to be released into solution and separated from protein contaminants. The final total RNA was further purified from contaminants by acid phenol and chloroform extraction and concentrated by isopropanol precipitation. Further purification was performed by ethanol precipitation and washing to remove any residual protein and contaminating salts.

[0101] The purity and quantity of total RNA extracted from tissue samples was determined by gel electrophoresis and indicated as the A260 / A280 ratio. About 3.5 mg of total ...

Embodiment 3

[0105] Prepare DNA for sequencing using standard DNA purification kits. DNA sequencing was performed using the dideoxy method developed by Sanger et al. (1977), which exploits the ability of DNA polymerases to incorporate 2',3'-dideoxynucleotides as substrates. A DNA polymerase is used to replicate a single-stranded DNA template by adding nucleotides to an extended strand. Chain extension occurs at the 3' end of the primer, the oligonucleotide that anneals to the template. The deoxynucleotides added to the extension products are selected by base pair matching with the template. The extension product grows by forming a phosphodiester bond between the 3'-hydroxyl group at the growing end of the primer and the 5'-phosphate group of the introduced deoxynucleotide. Growth takes a 5' to 3' direction. DNA polymerases are also capable of incorporating analogs of nucleotide bases. Upon incorporation of a dideoxynucleotide analogue at the 3' end of a growing chain, chain elongation ...

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Abstract

The invention is directed nucleic acids associated with sequences encoding motoneuronotrophic factors, which promote the survival, growth, proliferation, or maintenance of mammalian neurons, polypeptides encoded by the sequences, and assays for detecting the sequences.

Description

[0001] related application [0002] This application claims the benefit of US Provisional Application No. 60 / 518,581, filed November 7, 2003 (which is incorporated herein by reference in its entirety). field of invention [0003] The present invention relates to human genes encoding a specialized set of proteins that promote the growth, maintenance, survival and functional capacity of selected neuronal populations. Background of the invention [0004] Neuronotrophic factors (NTFs) are a group of specialized proteins whose function is to promote the survival, growth, maintenance and functional capacity of selected neuronal populations. Recent studies have demonstrated that neuronal death occurs in the nervous system of vertebrates during certain stages of growth and development. However, the addition of soluble neuronal trophic factors from the relevant target tissues helped to attenuate this neuronal de...

Claims

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Application Information

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IPC IPC(8): C07H21/02C07H21/04C12P21/06C12N5/00C12N15/00C12N15/63C07K14/475C12N
CPCC07K14/48C07K14/475
Inventor B·B·薛
Owner GENERVON BIOPHARM
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