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A carrier for inducing reversible immortalization of animal cells in vitro and its application

An animal body, circular vector technology, applied in the field of genetic engineering, can solve problems such as embryonic development arrest, cancerization, cell immortalization, etc.

Inactive Publication Date: 2016-04-27
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, immortalized cells or long-lived cells do not fully meet the requirements of animal cloning or cell therapy. Cells that stably express these "immortalized" genes for a long time cannot be used for somatic cell cloning or cell therapy, because cloning can easily lead to tumorigenesis or embryonic cell therapy. Developmental arrest
The existing technology mainly uses the method of continuously expressing the hTERT gene to prolong the lifespan of cells, but once the hTERT gene is transferred, its expression cannot be turned off, resulting in immortalization or canceration of the cells

Method used

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  • A carrier for inducing reversible immortalization of animal cells in vitro and its application
  • A carrier for inducing reversible immortalization of animal cells in vitro and its application
  • A carrier for inducing reversible immortalization of animal cells in vitro and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Example 1. Construction of Tet-on-mediated hTERT inducible expression vector (pTet-OnhTERT)

[0061] In this example, tetracycline (Tet-on)-mediated human telomerase gene (hTERT) inducible expression vector pTet-OnhTERT was constructed. The original plasmids used were pTet-OnAdvanced plasmid, pTRE-Tight plasmid, pIREShyg3 plasmid and pLPC-hTERT. Among them, the map of the pTet-OnAdvanced plasmid is as follows figure 1 shown; the map of the pTRE-Tight plasmid is shown in figure 2 As shown, the multiple cloning site (MCS) contains EcoRI and NheI recognition sequences from upstream to downstream; the map of pIREShyg3 plasmid is as follows image 3 As shown, there is an NheI recognition sequence at the multiple cloning site (MCS).

[0062] 1. Construction of recombinant vector pTet-OnhTERT

[0063] (1) Digest the pTet-OnAdvanced plasmid with XhoⅠ, and recover the 4.2kb fragment (containing the following element P CMV , rtTA-Advanced and SV40polyA, and ColE1ori and Amp...

Embodiment 2

[0076] Example 2, pTeton-hTERT mediates the reversible immortalization of sheep fetal fibroblasts

[0077] Cell culture medium: DMEM / F12 (product of Gibco, catalog number 12500062) + 10% (volume fraction) fetal bovine serum

[0078] Cell digestion solution: weigh 8.0g NaCl, 0.4g KCl, 1.0g glucose, NaHCO 3 , 0.35g, EDTA·tetrasodium salt 2.0g, trypsin 2.5g, with milliQH 2 O was adjusted to 1L. Sterilize by filtration and store at -20°C for later use.

[0079] Cell freezing medium: DMEM / F12+20% (volume fraction) fetal bovine serum + 10% (volume fraction) DMSO.

[0080] 1. Isolation and culture of primary sheep fetal fibroblasts

[0081] 1. Fetal tissue collection and cell separation:

[0082] The 70-day-old Small-tailed Han sheep fetus was collected by surgery, a small piece of fetal skin tissue was cut, put in sterile saline and brought back to the laboratory, and the tissue was cut to 1mm with scissors under aseptic operating conditions in an ultra-clean workbench 3 size,...

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Abstract

The invention discloses a carrier inducing reversible immortalization of in vitro animal cells and an application thereof. The carrier provided by the invention contains an escherichia coli replication initiation region, an ampicillin resistance gene, an expression cassette 1 and an expression cassette 2, wherein the expression cassette 1 comprises the following components from upstream to downstream in sequence: Ptight, hTERT, IVS, IRES, Hygr and SV40polyA; the expression cassette 2 comprises the following components from upstream to downstream in sequence: PCMV, rtTA-Advanced and SV40polyA; the expression cassette 1 is opposite to the expression cassette 2. Experiments prove that reversible regulation of lives of the animal cells can be achieved and the efficiency of in vitro cell genetic operation can be improved by utilizing the annular carrier provided by the invention. The carrier is of great significance in providing safe cell materials for animal genetic modification or medical cell therapy and the like.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a carrier for inducing reversible immortalization of animal cells in vitro and its application. Background technique [0002] Animal cells cultured in vitro have important application value in biological research, animal cloning, transgenic and cell therapy, but the proliferation of primary cells isolated from animal tissues is limited, which is not conducive to long-term culture in vitro, especially for genetic modification and tissue engineering and other long-term training processes. The lifespan of primary cultured somatic cells can be extended by expressing certain genes, that is, the ability of immortalization can be obtained, among which the human telomerase catalytic subunit gene (hTERT) is one of the commonly used genes. It has been confirmed that expressing human telomerase catalytic subunit gene (hTERT) in cells can make cells obtain functional telomerase, prevent telo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/64C12N5/10
Inventor 侯健张娜安晓荣
Owner CHINA AGRI UNIV
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