Construction method of mouse model with conditional over-expression of HPV E7 gene at H11 locus

A technology of mouse model and construction method, which is applied in the fields of molecular biology and biomedicine, can solve the problems of high time and money costs, and achieve the effects of easy targeting, increased probability of homologous recombination repair, and high gene editing efficiency

Pending Publication Date: 2020-03-27
SHANGHAI TONEKER BIOTECH
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Problems solved by technology

Establishing mouse strains in this way

Method used

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  • Construction method of mouse model with conditional over-expression of HPV E7 gene at H11 locus
  • Construction method of mouse model with conditional over-expression of HPV E7 gene at H11 locus
  • Construction method of mouse model with conditional over-expression of HPV E7 gene at H11 locus

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specific Embodiment

[0064] Specific examples: Construction of conditionally overexpressed HPV16 E7 animal model at the H11 site

[0065] Using CRISPR-Cas9 gene editing technology to knock in the HPV16 E7 gene sequence at the H11 site of the mouse model, the conditional overexpression of HPV16 E7 can be achieved, so as to establish the conditional overexpression of HPV16 E7 mouse model. Including the following steps:

[0066] (1) sgRNA design and evaluation of off-target effects

[0067] sgRNA design for conditional overexpression of HPV16 E7 at the H11 locus of C57BL / 6 mice, and off-target effect evaluation of candidate sgRNAs, such as figure 1 As shown, the screened sgRNA (matching forward strand of gene):

[0068] GAACACUAGUGCACUUAUCCUGG.

[0069] (2) co-incubating the obtained sgRNA with the Cas9 protein to prepare a Cas9 / sgRNA mixture;

[0070] (3) Gene editing strategy and targeting vector construction

[0071] The overall strategy of gene editing is as follows: figure 2 As shown, in...

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Abstract

The invention provides a construction method of a mouse model with conditional over-expression of HPV E7 gene at H11 locus. The construction method of the mouse model with the conditional over-expression of the HPV E7 gene at the H11 locus comprises the following steps: designing and obtaining sgRNA; (2) preparing a Cas9/sgRNA mixture; (3) constructing a targeting vector; (4) co-injecting the targeting vector and the Cas9/sgRNA mixture into fertilized eggs of a mouse so as to subsequently obtain F0 generation mice; and (5) obtaining F1 generation mice. The construction method of the mouse model with the conditional over-expression of the HPV E7 gene at the H11 locus solves the problem that "nude mice are not suitable for studying role of HPV E7 in HPV E7-mediated processes of tumor microenvironment immune regulation, immune escape and the like for the nude mice lack competent immune systems" in the prior art. The mouse model provided by the invention has a competent immune system, so that the mouse model is suitable for all researches on interaction between the HPV E7 and the immune system.

Description

technical field [0001] The invention relates to a method for constructing a mouse model for conditionally overexpressing HPV E7 gene at the H11 site, and belongs to the technical fields of molecular biology and biomedicine. Background technique [0002] Cervical cancer is one of the most common malignant tumors in women, and its incidence is second only to breast cancer among gynecological malignancies. In recent years, the incidence of cervical cancer in my country has been on the rise, and tends to be younger. Human papillomavirus (HPV) belongs to the Papillomavirus A genus of the Papovaviridae family. It is a spherical DNA virus that specifically infects epithelial cells. At present, more than 200 HPV subtypes have been identified, which can be divided into high-risk HPV (hrHPV) and low-risk HPV (lrHPV) according to their carcinogenicity. Persistent hrHPV infection is the main cause of high-grade cervical lesions and cervical cancer, and more than 90% of cervical cancer...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90A01K67/027
CPCC12N15/85C12N15/907A01K67/0278A01K2217/072A01K2227/105A01K2267/0331C12N2999/007
Inventor 袁奕马烽鲁京李昂何胜祥
Owner SHANGHAI TONEKER BIOTECH
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