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Helper plasmid for high efficiency building of transgenic mouse model and construction method thereof

A technology of transgenic mice and auxiliary plasmids, applied in the field of genetic engineering, can solve problems such as low efficiency, and achieve the effect of simple operation method, efficient construction, and improved efficiency

Active Publication Date: 2015-10-14
重庆高圣生物医药有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is first to solve the problem of low efficiency of exogenous gene integration into the recipient genome in the process of constructing a transgenic mouse model of human disease using gene targeting technology, and to provide an auxiliary plasmid for efficiently establishing a transgenic mouse model

Method used

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  • Helper plasmid for high efficiency building of transgenic mouse model and construction method thereof
  • Helper plasmid for high efficiency building of transgenic mouse model and construction method thereof
  • Helper plasmid for high efficiency building of transgenic mouse model and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 The helper plasmid mediates the insertion of pDsRed2-N1 plasmid into the mouse genome

[0023] (1) Construction of helper plasmid

[0024] The pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (Addgene plasmid ID: 42230, hereinafter referred to as pSpCas9(BB)) was digested with BbSI, and after 1 hour in a water bath at 37°C, electrophoresed on 1% agarose to recover the digested product ( TAKARA gel recovery kit).

[0025] The enzyme digestion system is as follows:

[0026]

[0027] Use the online tool ZiFiT Targeter version 4.0 to design oligonucleotides targeting mouse genome-specific target site sequences, specifically:

[0028] Specific target site sequence: 5'-GGGGGGGAGCCAGCGCGCTG-3' (SEQ ID NO.1);

[0029]The corresponding oligonucleotide pair is: Oligo (+): 5'-caccGGGGGGGAGCCAGCGCGCTG-3' (SEQ ID NO.2);

[0030] Oligo (-): 5'-aaacCAGCGCGCTGGCTCCCCCCC-3' (SEQ ID NO. 3).

[0031] Anneal the two oligonucleotides to form a short double-stranded DNA with sticky...

Embodiment 2

[0048] Example 2 The helper plasmid mediates the insertion of pEGFP-N1 plasmid into the mouse genome

[0049] The helper plasmid pSpCas9(32T) was obtained according to the method in step (1) of Example 1. The linearized pEGFP-N1 plasmid (Clontech, specifically refer to the Clontech instructions) and the pSpCas9 (32T) helper plasmid were co-transfected into a mouse liver cancer cell line (Shanghai Cell Bank, Chinese Academy of Sciences) by electroporation (see step 1 in Example 1 for specific operations ( 3)), under the same conditions, only the linearized pEGFP-N1 plasmid, the pSpCas9(32T) helper plasmid, and no plasmid were transfected as controls.

[0050] 24 hours after transfection, adherent cells were digested with trypsin, collected by centrifugation, washed twice with PBS, and then transiently transfected GFP-positive cells were sorted by flow cytometry. The results showed that only transfected pEGFP-N1 plasmid ( image 3 C) and pEGFP-N1+pSpCas9 (32T) recombinant plas...

Embodiment 3

[0052] Example 3 Preparation of transgenic mouse tumor model by helper plasmid mediating pbabe-c-mycT58A+HRasG12V plasmid

[0053] The helper plasmid pSpCas9(32T) was obtained according to the method in step (1) of Example 1. The linearized pbabe-c-mycT58A+HRasG12V plasmid (Addgene plasma ID: 11130, see specifically: Kendall SD, Linardic CM, Adam SJ, Counter CM.A network of genetic events sufficient to convert normal human cells to a tumorigenic state[ J].Cancer Res.2005, 65(21):9824-8) and pSpCas9(32T) helper plasmid co-transfect mouse fertilized oocytes by microinjection (see Chi Jun, Kuang Ying et al., DNA Fragment Size The effect on the integration efficiency of the preparation of transgenic mice [J], Experimental Animals and Comparative Medicine, 2012, 32(2), 116-119), and only linearized pbabe-c-mycT58A+HRasG12V plasmids were transfected under the same conditions , pSpCas9(32T) helper plasmid, and no transfection of any plasmid were used as controls, and 100 fertilized ...

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Abstract

The invention discloses a helper plasmid for high efficiency building of a transgenic mouse model and a construction method thereof. When the helper plasmid and an exogenous gene cotransfect a mouse recipient cell, the helper plasmid can form 32 gaps on a mouse genome specific target site sequence and the 32 gaps provide 32 integration hot spots for the exogenous gene so that efficiency of integration of the exogenous gene in the mouse genome is substantially improved. The construction method is simple, has a low cost, and can be carried out in a common laboratory. The helper plasmid can directly cotransfect the mouse recipient cell with the different exogenous genes. The construction method can be operated simply. The helper plasmid can effectively improve efficiency of stable transgenic cell line expression in the mouse, realizes high efficiency construction of various human disease transgenic mouse models and is conducive to research on human disease pathogenesis and development of gene treatment.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an auxiliary plasmid for efficiently establishing a transgenic mouse model and a construction method thereof. Background technique [0002] The development of human diseases is very complicated. Using humans as experimental objects to deeply explore the mechanism of disease occurrence and promote the development of medicine has come slowly. The accumulated clinical experience is not only limited in time and space, but also many experiments are morally unreasonable. and methods are also limited. With the help of indirect research on animal models, factors that are impossible or difficult to exclude under natural conditions can be consciously changed, so as to observe the experimental results of the model more accurately and conduct comparative studies with human diseases, which will help make it easier and more effective. Effectively understand the occurre...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66
Inventor 周勇温平申友锋唐秋月何燕刘兰兰向垚艮李建均
Owner 重庆高圣生物医药有限责任公司
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