Allele double knockout targeting vector system and construction method thereof

A technology for targeting vectors and alleles, applied in the field of double allele knockout targeting vector systems and their construction, can solve the problems of few surviving cells, ineffective strategy, and long cycle, and achieve the effect of shortening the experimental time and improving the efficiency.

Inactive Publication Date: 2011-06-15
NORTHWEST A & F UNIV
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  • Description
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  • Application Information

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Problems solved by technology

However, this method takes a long time, and there are very few surviving cells under the pressure of high concentrations of antibiotics, and if the single-copy gene knockout cells develop resistance to the maximum concentration of antibiotics, the strategy will fail
Furthermore, it is difficult to determine whether the cell death is

Method used

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  • Allele double knockout targeting vector system and construction method thereof
  • Allele double knockout targeting vector system and construction method thereof
  • Allele double knockout targeting vector system and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0051] Materials and Reagents

[0052] (1) Strains and cells

[0053] DH5α (preserved in our laboratory), mouse C2C12 (isolated in our laboratory)

[0054] (2) Plasmid

[0055] pIRES-neo(CLONTECH), pEGFP-C1(CLONTECH), pDsRed-N1(CLONTECH), pBS246(GIBCO / BRL), pBS185(GIBCO / BRL), pCEP4(Invitrogen), pORF-HSV1tk(Invivogen), pMD20-T (TaKaRa)

[0056] (3) Reagents

[0057] Various endonucleases, T4 ligase, KLENOW, CIP, etc. were purchased from MBI; G418 (GIBCO), guanosine (GAC) (Sigma), hygromycin (Roche), DMEM dry powder (high sugar) (GIBCO ), Fetal Bovine Serum (GIBCO), DNA Fragment Rapid Recovery Kit (BioDev-tech), Small Plasmid Extraction Kit (Vigrous)

[0058] (4) Primers and sequences

[0059]

[0060] Method steps

[0061] 1) Construction of intermediate vector pIRES-Neo-GFP

[0062] pEGFP-C1 was single-digested with Nhe Ⅰ, single-digested with Bgl Ⅱ after Klenow filling, and pIRES-neo was double-digested with EcoR Ⅴ and BamHI at the same time, the EGFP fragment and ...

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Abstract

The invention relates to an allele double knockout targeting vector system and a construction method thereof. The system of the invention consists of two complementary vectors, namely pGT-V1 and pGT-V2, wherein each vector contains two in phase LoxP elements which contain a positive selection marker gene Neo/GFP and Hyg/RFP respectively; and the outside of each vector contains a negative selection marker gene TK. Meanwhile, two 8-basic group multiple cloning sites are designed and arranged between the two LoxP elements and the negative selection marker for the insertion of the homology arm. By adopting the constructed targeting vector system of the invention, two complementary targeting vectors are cotransfected into the recipient cell; through the selection of drug and fluorescent double-selection marker, the genetic modification or knockout of the two alleles of the target gene can be realized once; and the interaction of the transfected Cre enzyme and the LoxP elements can be utilized to remove the selection marker gene integrated with the genome, the time of obtaining the homozygous knockout target cell can be shortened, the safety of the transgenic animal can be increased and a valuable technology platform is provided to develop the animal transgenic researches.

Description

[0001] Technical Field: Genetic Engineering technical background: [0002] Research on transgenic animals is now a hot topic in the scientific community. Through transgenic technology, people can break the isolation between species to obtain economic animals with excellent traits, obtain various biological materials and medicinal proteins through the production of bioreactors, break immune rejection for organ transplantation, and establish animal models of various human diseases As well as analysis to study specific gene function. [0003] However, the existing transgenic technology still has drawbacks. The biggest problem is the position effect caused by the random insertion of foreign genes, that is, the random insertion of foreign genes into genes related to important life activities of cells causes their abnormal expression. In addition, if the gene of interest is randomly inserted into chromatin to generate higher-order structures, or inserted into a heterochromatin regi...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/65C12N15/66
Inventor 王华岩韩翠芹李军华
Owner NORTHWEST A & F UNIV
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