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Method for efficiently establishing pure line gene knock-out mouse model

A technology of transgenic and suicide genes, applied in the field of transgenic technology, can solve the problem of rarely using mice

Inactive Publication Date: 2005-02-16
SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the various health problems common in cloned animals, this method is rarely used in gene knockout mice

Method used

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  • Method for efficiently establishing pure line gene knock-out mouse model
  • Method for efficiently establishing pure line gene knock-out mouse model
  • Method for efficiently establishing pure line gene knock-out mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Construction of IAP-TK plasmid

[0051] The primers IAPP1 and IAPP2 were synthesized, and the mouse cell genomic DNA was used as a template to amplify the fragment IAPP1P2 containing the IAP3' end; the primers IAPP3 and IAPP4 were synthesized to amplify the fragment IAPP3P4 containing the IAP5' end.

[0052] Primer

[0053] The IAPP1P2 fragment digested by BamHI and SalI was cloned into the SalI-BamHI site of pGL3-basic (Promega Company), and then the full-length hsv-TK gene (this fragment was obtained from pPNT (transgene available from University of Michigan) Animal Model Center)) was cloned into the XbaI-XhoI site, and finally the IAPP3P4 fragment was cloned into the KpnI-SmaI site to obtain the construction of the transgenic plasmid IAP-TK ( figure 1 ).

Embodiment 2

[0055] Introduction of hsv-TK into fertilized eggs of mice by microinjection and production of transgenic positive mice

[0056] After the transgenic plasmid IAP-TK was linearized with SalI and NotI, the fragments containing IAP and hsv-TK genes were recovered.

[0057] Linearized DNA (about 500 copies) was injected into the male pronucleus of mouse fertilized eggs, and the injected fertilized eggs were transplanted into pseudopregnant mice. About 20 days later, the mice were born. After 3 weeks of birth, the tail was cut to extract DNA, and PCR (hsv-TK gene-specific primers 5'-CCCCTTCTTCGCTGGTACGAGGAG-3'(SEQ ID NO: 5) and 5'-AAGCGCGTGGAGTTGACCTGAAGT-3'(SEQ ID NO: 6)) detection Analyze for transgene integration.

[0058] The result is as figure 2 shown. A total of 11 transgenic positive mice were obtained, and these mice were bred to establish lines, and a total of 3 transgenic mouse lines were established.

Embodiment 3

[0060] Expression of Transgene in Mouse Testis

[0061] In order to detect whether the IAP-TK fusion gene is expressed in mice, the total RNA of mouse salivary glands was extracted with Trizol reagent according to conventional methods. According to the Takara RNA PCR Kit (Ver2.1) manual, reverse transcription was performed, and then PCR amplification was performed to detect the expression of hsv-TK. The total RNA of transgenic mouse testis, epididymis, coagulation gland, seminal vesicle gland, heart, liver, lung, spleen, kidney, muscle, brain and other tissues were analyzed.

[0062] The results showed that hsv-TK was only expressed in testis tissue ( image 3 ).

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Abstract

The invention provides a kind of transgenic inhuman mammal by producing klone gene without mouse model. Roll outside self murder gene into the mammal. The expression box contains promoter, self murder gene relation with promoter and termination codon. The natural masculine gene will express in testes organize after transgenic animal builds system. The transgenic animal can provide blastosphere for eliminating gene technic to improve the efficiency of the technic and to solve the problem that mouse genetic background is impure.

Description

technical field [0001] The invention relates to the field of transgenic technology. More specifically, it relates to a pure line gene knockout mouse model, a method for establishing the pure line gene knockout mouse model, and uses of the pure line gene knockout mouse model. Background technique [0002] Since the advent of gene knockout technology, it has been widely used in life science research. The gene knockout technology itself is also constantly developing, and conditional gene knockout techniques such as developmental stage specificity, tissue type specificity and drug induction have emerged, which greatly facilitate the detailed study of gene function. Gene knockout technology has become one of the most important means of gene function research. [0003] Currently, there are three main ways to obtain gene knockout mice from homologous recombination cells. [0004] One is to carry out gene targeting at the ES cell level to obtain homologous recombination ES cell c...

Claims

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Application Information

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IPC IPC(8): A01K67/027C12N5/10C12N15/12C12N15/52C12N15/87
Inventor 王铸钢赵旭东任维华王龙孔辉
Owner SHANGHAI BIOMODEL ORGANISM SCI & TECH DEV
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