Porcine somatic cell mutagensis method

A technology of somatic cells and cells, applied in the field of cell mutagenesis, can solve the problems that have not been reported in the literature, and achieve the effects of simple and convenient operation, short induction period and stable growth state

Inactive Publication Date: 2011-09-07
ANHUI AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

However, up to now, there have been no literature reports on the induction technology of using lentivirus to induce porcine somatic cells into stem cells at home and abroad.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0017] 1 Materials and reagents:

[0018] Isolation and cultivation of fetal pig fibroblasts: a tissue block culture method was used to establish a fetal pig fibroblast cell line, and the culture medium of fetal pig fibroblasts was high-sugar DMEM (Hyclone) containing 10% fetal bovine serum (Hyclone).

[0019] Establishment of mouse embryo fibroblasts (MEFs) line: Fetal mice of ICR strain were taken at 12.5 days of pregnancy, and mouse embryo fibroblasts were isolated and cultured by trypsinization method after removing the head, limbs and viscera. The culture medium of fetal mouse fibroblasts is high glucose DMEM (Hyclone) containing 10% newborn bovine serum (Sijiqing).

[0020] Preparation of mouse fetal fibroblast feeder layer: select 2-4 generation mouse embryonic fibroblasts that cover the bottom of the dish, treat with 10 μg / ml mitomycin C (Sigma) for 2.5 hours, and digest with conventional trypsin Inoculate into a petri dish covered with 0.1% gelatin at a cell density ...

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Abstract

The invention discloses a porcine somatic cell mutagensis method. Porcine embryonic fibroblasts are infected and acted a plurality of times by using slow viruses carrying enhanced green fluorescent protein markers; the original fibrous growth forms of the porcine embryonic fibroblasts are changed step by step under a growing environment condition of a stem cell culture solution to form nest-like cell colonies; separated, cultured and sub-cultured cell colonies have clear edges and boundaries, stable growth states and normal karyotypes; alkaline phosphatase is expressed to be positive; immunocytochemical detection shows that Oct4, Nanog and SSEA-1 proteins are expressed to be positive; teratoma comprising three embryonic layers is formed by differentiation in vivo; and the result proves that the mutagenic porcine somatic cells have the stem cell properties under the action of the slow viruses.

Description

[technical field] [0001] The invention relates to the technical field of cell mutagenesis, in particular to a method for mutagenesis of pig somatic cells. [Background technique] [0002] As a kind of recombinant retroviral vector, lentiviral vector has the advantages of convenience, quickness, high efficiency and stable transfection in transgenic production. It has become an important gene transfer tool and is widely used in the field of gene transfer into cell molecular biology research. . Lentivirus is a member of the retrovirus family. The virus can use its own components to integrate foreign genes into the host cell genome, so that foreign genes can be stably expressed with the division, proliferation and passage of host cells. In 2006, for the first time, humans used retroviruses to mediate the expression of Oct4, Sox2, Klf4 and c-Myc in mouse skin fibroblasts, and the mouse fibroblasts expressing these exogenous genes presented mouse embryonic stem cells (Embryonic st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/10C12N7/01C12N15/867
Inventor 曹鸿国章孝荣刘亚张运海陶勇方富贵殷慧群孙雪萍薛奕杰张卫琴黄伟玲陈涛李运生任春环张子军丁建平刘洪瑜刘旭光王力生
Owner ANHUI AGRICULTURAL UNIVERSITY
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