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Construction method of glial cell line-derived neurotrophic factor gene-modified embryo neural stem cell

A technology of neurotrophic factors and neural stem cells, applied to cells modified by introducing foreign genetic material, genetic engineering, plant genetic improvement, etc., can solve problems such as little effect and limited expression of factors

Inactive Publication Date: 2013-07-24
孙勇
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Problems solved by technology

Although NSCs can produce a series of neurotrophic factors such as GDNF, BDNF, NGF, NT-3, NT-4 / 5 [57] , but these factors have limited expression and thus little effect

Method used

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  • Construction method of glial cell line-derived neurotrophic factor gene-modified embryo neural stem cell
  • Construction method of glial cell line-derived neurotrophic factor gene-modified embryo neural stem cell
  • Construction method of glial cell line-derived neurotrophic factor gene-modified embryo neural stem cell

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Embodiment Construction

[0029] This specific embodiment illustrates the construction of embryonic neural stem cells genetically modified with glial cell-derived neurotrophic factors of the present invention. Follow the steps below:

[0030] (1) Transform Escherichia coli with the plasmid, and inoculate TG1 Escherichia coli in LB medium to prepare competent Escherichia coli. At the same time, the plasmid pSK / GDNF containing the target gene glial cell-derived neurotrophic factor gene (GDNF) and the retroviral vector plasmid pLNCX2 were added to the competent cell suspension, and transferred to a 100mm LB plate containing Amp. , After inverting the plate and incubating at 37°C for 12-16 hours, colonies may appear. Pick positive colonies for plasmid extraction and purification.

[0031] (2) Digest the plasmid with a restriction endonuclease, add ddH to a disposable 200 μl Eppendorf tube in sequence 2 O 1 μl, Buffer 2 μl, DNA 15 μl (purified pSK / GDNF plasmid or pLNCX2 plasmid), HindIII and NotI 1 μl ea...

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Abstract

The invention relates to a biotechnology and especially relates to a construction method of a glial cell line-derived neurotrophic factor gene-modified embryo neural stem cell. The construction method comprises the following steps of 1, plasmid transformation, 2, plasmid digestion purification, 3, target gene and vector cloning, 4, recombinant plasmid packaging, 5, recombinant plasmid identification, 6, virus titer determination, 7, stem cell culture, 8, virus infection of the stem cells, 9, transgenic stem cell identification, 10, transgenic stem cell target-protein content determination, and 11, transgenic stem cell biological-activity detection. The glial cell line-derived neurotrophic factor gene-modified embryo neural stem cell has strong effects of promoting survival of dopaminergic neurons and retains characteristics of the original neural stem cell. The glial cell line-derived neurotrophic factor gene-modified embryo neural stem cell has good effects of treating motor neuron damage diseases and neuronal degeneration diseases.

Description

technical field [0001] The invention relates to a biological technology, in particular to the construction of embryonic neural stem cells genetically modified by glial cell-derived neurotrophic factors. technical background [0002] In 1988, Rousselet et al. discovered that the conditioned medium of glial cells had neurotrophic effects. In 1993, Chinese-American scientist Lin et al. purified the protein with neurotrophic effects from the conditioned medium of glioma cells and cloned its gene. And named it glial cell line-derived neurotrophic factor (GDNF). Subsequent numerous studies have shown that GDNF has a strong pro-survival effect not only on dopaminergic neurons, but also on motor neurons, norepinephrine neurons, peripheral sensory neurons, and sympathetic neurons ; and GDNF also has a good nutritional effect on developing motor neurons. The effect of GDNF on motor neurons is the most active one among all the neurotrophic factors found so far, which can promote the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N7/01G01N33/68
Inventor 孙勇
Owner 孙勇
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