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Novel use of gene DCF1 related to neural stem cell differentiation

A neural stem cell and gene technology, applied in genetic engineering, plant genetic improvement, application, etc., can solve problems such as unreported functions

Inactive Publication Date: 2008-10-08
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In neural stem cells, the function of dendritic cell factor 1 (DCF1) gene has not been reported

Method used

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  • Novel use of gene DCF1 related to neural stem cell differentiation
  • Novel use of gene DCF1 related to neural stem cell differentiation
  • Novel use of gene DCF1 related to neural stem cell differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1: Construction and identification of bait plasmid pGBKT7-DCF1

[0015] The upstream and downstream primers of DCF1 were designed using Premier 5.0 software, and EcoRI and SalI restriction sites were introduced into the upstream and downstream primers, respectively. This pair of primers was synthesized by Shanghai Sangong. Using mouse brain cDNA as a template, the full-length cDNA of DCF1 was amplified with synthetic upstream and downstream primers, digested with EcoR I and SalI, and connected to the pGBKT7 vector that was also digested with EcoR I and SalI. Enzyme digestion, PCR identification, correct sequencing, see figure 1 . AH109(pGBKT7-DCF1) cannot grow in SD / -Trp / -His, and in the β-galactosidase activity filter copy analysis, the colonies remain white within 8 hours without turning blue, see figure 2 . It suggested that the pGBKT7-DCF1 plasmid did not activate the expression of the reporter gene itself. can be used in subsequent experiments.

Embodiment 2

[0016] Example 2: Screening of mouse brain cDNA library

[0017] The purified mouse brain cDNA library plasmid and pGBKT7-DCF1 bait plasmid were co-transformed into yeast cell AH109, ​​and a total of 5.2X10 7 a transformant. Collect the yeast from the plate and titrate, the titer is 0.59X10 8 . Take 1.57X10 8 A total of 1820 clones were grown from 1 yeast strain on the SD / -Trp-Leu-His plate; these clones were picked onto the SD / -Trp-Leu-His-Ade plate with a toothpick to obtain 105 single clones. The expression of these 105 monoclonal β-galactosidase genes was detected by fliter-assay (blue and white screening), the results are as follows image 3 As shown, a total of 20 clones were blue (that is, expressed β-galactosidase gene), which were positive clones. The plasmids containing the pACT2 vector were extracted from the 20 positive clones obtained above, which were respectively electrotransformed into Escherichia coli DH5α for amplification, and the plasmids were extracte...

Embodiment 3

[0018] Example 3: Fluorescence colocalization verification of the interaction between DCF1 and ATP1B1

[0019] The upstream and downstream primers of ATPase, Na+ / K+transporting, and beta 1 polypeptide were designed using Premier 5.0 software, and EcoRI and BamHI restriction sites were introduced into the upstream and downstream primers, respectively. This pair of primers was synthesized by Shanghai Sangong. Using mouse brain cDNA as a template, use synthetic upstream and downstream primers to amplify the full-length cDNA of ATPase, Na+ / K+transporting, beta 1 polypeptide, EcoRI and BamHI double digestion, and connect to the same EcoRI and BamHI double digestion In the pDSRED2-C1 vector, enzyme digestion and PCR identification are correct, see Figure 4 . pDSRED2-ATP1B1 and pEGFP-DCF1 (for their construction methods, see Lei Wang, Jiao Wang, YiliuWu, Jie Wu, Shuya Pang, Rong Pan, Tieqiao Wen (2008) A Novel Function of dcflDuring the Differentiation of Neural Stem Cells In vitr...

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Abstract

The invention relates to a new usage of a neural stem cell differentiating related gene DCF1. The invention sieves protein ATP1B1 interacted with DCF1 through utilizing a yeast-two hybrid technology and is positioned altogether by using cell fluorescence, and the interaction is confirmed through immunity and precipitation. According to a literature report, the interaction between ATP1B1 and BACE1 exists, but BACE1 is an essential gene of the development of AD (Alzheimer disease). After DCF1 is muted through a RNAi method, the expression of ATP1B1 and BACE1 also changes remarkably the results prove that the expression of BACE1 can be adjusted and controlled through certain signal passages of DCF1, certain relation exists in the occurrence and the development of DCF1 and AD, which is possible to be a new drug effect target spot of AD treatment, the interaction of DCF1 and ATP1B1 is found, and the invention has a vital significance to a signal conducting mechanism during the understanding process of the development and the progression of AD.

Description

technical field [0001] The invention relates to a new application of a gene DCF1 related to neural stem cell differentiation. Background of the invention [0002] Neural stem cells (neural stem cells, NSCs) can self-replicate and have multi-lineage differentiation potential, and have important application value in the treatment of central nervous system injury and degenerative diseases. Transplanting neural stem cells into the damaged area can differentiate into specific nerve cells in the body to complete specific neural functions. However, there are many technical difficulties in neural stem cell transplantation, among which the ability of neural stem cells to differentiate and participate in neural network reconstruction after transplantation has become the focus of attention. To clarify the differentiation mechanism of neural stem cells has important theoretical significance and practical value for solving the above problems. [0003] In neural stem cells, the function...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12Q1/68G01N33/53G01N21/64
Inventor 文铁桥吴翌鎏钱仁红
Owner SHANGHAI UNIV
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