Construction method of mouse model for conditional overexpression of HPV E6 gene at ROSA26 site

A mouse model and construction method technology, applied in the biological field, can solve the problems of easy loss of phenotype, irreproducible experimental results, high time and money costs, etc., and achieve the effects of high gene editing efficiency, precise experimental operation, and easy targeting

Pending Publication Date: 2020-04-17
SHANGHAI TONEKER BIOTECH
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Problems solved by technology

Moreover, due to the dilution of the copy number of the transgene and gene silencing after the passage of the transgenic mice, the phenotype of the same strain is also easily lost during the passage, resulting in unrepeatable experimental results.
Establishing mouse strains in this way requires a high cost of time and money

Method used

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  • Construction method of mouse model for conditional overexpression of HPV E6 gene at ROSA26 site
  • Construction method of mouse model for conditional overexpression of HPV E6 gene at ROSA26 site
  • Construction method of mouse model for conditional overexpression of HPV E6 gene at ROSA26 site

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specific Embodiment

[0063] Specific example: Construction of conditional overexpression HPV16 E6 animal model at ROSA26 site

[0064] Using CRISPR-Cas9 gene editing technology to knock in the HPV16 E6 gene sequence at the ROSA26 site of the mouse model, the conditional overexpression of HPV16 E6 can be achieved, so as to establish the conditional overexpression of HPV16 E6 mouse model. Including the following steps:

[0065] (1) sgRNA design and evaluation of off-target effects

[0066] sgRNA design for conditional overexpression of HPV16 E6 at the ROSA26 locus in C57BL / 6 mice, and off-target effect evaluation of candidate sgRNAs, such as figure 2 shown. Screening to get sgRNA(matching forward strand ofgene):

[0067] CUCCAGUCUUUCUAGAAGAUGGG.

[0068] (2) co-incubating the obtained gRNA with the Cas9 protein to prepare a Cas9 / sgRNA mixture;

[0069] (3) Gene editing strategy and targeting vector construction

[0070] The overall strategy of gene editing is as follows: figure 1 As shown, ...

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Abstract

The invention provides a construction method of a mouse model for conditional overexpression of an HPV E6 gene at an ROSA26 site, which comprises the following steps: designing and obtaining sgRNA according to the sequence of the HPV E6 gene, and preparing a Cas9 / sgRNA mixture; constructing a targeting vector, carrying out microinjection and obtaining an F0-generation mouse; and obtaining an F1-generation mouse. The method has the advantages of high editing efficiency and targeting ability, easy targeting and more accurate and efficient experimental operation.

Description

technical field [0001] The invention relates to a method for constructing a mouse model for conditionally overexpressing HPV E6 gene at the ROSA26 site, and belongs to the field of biotechnology. Background technique [0002] Cervical cancer ranks first in the malignant tumors of the female reproductive tract. It is one of the more common cancers and poses a great threat to women's health. In recent years, new cases of cervical cancer in my country are on the rise, and they are getting younger. In recent years, a large number of studies have concluded that canceration is a continuous process. The relationship between human papillomavirus (HPV) infection and cervical cancer has been confirmed. Almost all cervical cancers (>99%) are related to high-risk HPV (hrHPV). However, the molecular mechanism of HPV infection leading to cervical cancer is still unclear. Although the proportion of HPV-infected population is large, more than 80% of the transient positive carriers will...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90A01K67/027
CPCC12N15/85C12N15/907A01K67/0275A01K2217/07A01K2227/105A01K2267/0337
Inventor 袁奕马烽鲁京李昂何胜祥
Owner SHANGHAI TONEKER BIOTECH
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