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Method of preparing stem cell line for excreting insulin by using slow virus vectors of coding multiple exogenous gene

A technology of exogenous genes and stem cell lines, applied to cells modified by introducing foreign genetic materials, using vectors to introduce foreign genetic materials, recombinant DNA technology, etc., can solve problems such as inability to achieve stable cell transfection, and improve insulin expression level, high transfection efficiency, and simple construction method

Inactive Publication Date: 2008-01-09
CHINA JAPAN FRIENDSHIP HOSPITAL
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Problems solved by technology

[0005] In the past, adenovirus was used to simultaneously transfect the three insulin-regulated genes of MafA, NeuroD1 and PDX-1, and the adenovirus vector could not achieve stable transfection of cells

Method used

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  • Method of preparing stem cell line for excreting insulin by using slow virus vectors of coding multiple exogenous gene
  • Method of preparing stem cell line for excreting insulin by using slow virus vectors of coding multiple exogenous gene

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Embodiment Construction

[0027] A method for preparing an insulin-secreting stem cell line using a lentiviral vector encoding multiple foreign genes, comprising the steps of:

[0028] 1) Construction of lentiviral vectors encoding Neomycin, MafA, NeuroD and PDX-1 genes

[0029] First, the lentiviral vector plasmid pWPTS-GFP was double digested with BamH1 and Sal I enzymes, and after releasing GFP, the vector pWPTS fragment was recovered; then the plasmids containing the genes of Neomycin, MafA, NeuroD1 and PDX-1 were passed through BstB1 and After digestion with Bgl II, Xbal I and Not I, EcoR I and Xbal I, and EcoRI restriction endonucleases, use 0.7% agarose gel electrophoresis analysis to recover the target gene fragments; Ligated into the vector pWPTS fragment, constructed into lentiviral vectors encoding Neomycin, MafA, NeuroD and PDX-1 genes respectively;

[0030] 2) Packaging of lentiviral particles

[0031] ①Amplification of packaging plasmid:

[0032] The lentiviral packaging plasmids pMD2G...

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Abstract

Production of secretory insulin dry cell system by encoded various exogenous gene slow virus carrier is carried out by copying pWPST, cloning various insulin regulating genes to the carrier, transfecting 293T cell, packing, collecting virus grains, concentrating and transfecting human embryo pancreas dry cell. It's simple, efficient and practical, it can obtain one, two or three kinds of exogenous gene cells, which are in different expression and meet different needs.

Description

technical field [0001] The invention relates to a method for preparing insulin-secreting stem cell lines, in particular to a method for preparing insulin-secreting stem cell lines using lentiviral vectors encoding multiple foreign genes. Background technique [0002] Diabetes is one of the major diseases that seriously endanger human health, and has become the third major disease after cardiovascular diseases and tumors. In recent years, the incidence of diabetes has been increasing, and the onset is getting younger. According to literature reports: the diabetic patient of the whole world has surpassed 230 million, and with the speed increase of 2 million people every year. [0003] Diabetes patients in my country have exceeded 40 million and are expected to reach 60 million by 2010. Diabetic patients have disorders of glucose metabolism due to insulin production disorders or insulin failure to exert physiological functions, resulting in microvascular disease, causing a se...

Claims

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Application Information

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IPC IPC(8): C12N15/86C12N5/10
Inventor 门秀丽张文健娄晋宁
Owner CHINA JAPAN FRIENDSHIP HOSPITAL
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