Construction method for mouse model realizing conditional site-specific double-overexpression of HPV E6/E7 genes

A technology of mouse model and construction method, applied in the biological field, can solve the problems of genome rearrangement, non-expression, unfavorable control of gene expression level and function research, etc.

Pending Publication Date: 2020-04-21
SHANGHAI TONEKER BIOTECH
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Such mice are generally prepared by injecting pronuclei of fertilized eggs with plasmids. The integration is random, sometimes at a single site, and sometimes at multiple sites on different chromosomes. The randomness may cause Genome rearrangement, translocation deletion, and insertion of exogenous genes may destroy endogenous genes in mice; in addition, the number of integrated copies is not fixed, and multi-copy tandem integration may lead to low or no expression of exogenous genes, which is not conducive to the control of gene expression. Expression level and function studies

Method used

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  • Construction method for mouse model realizing conditional site-specific double-overexpression of HPV E6/E7 genes
  • Construction method for mouse model realizing conditional site-specific double-overexpression of HPV E6/E7 genes
  • Construction method for mouse model realizing conditional site-specific double-overexpression of HPV E6/E7 genes

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0093] Specific example 1: Carry out the construction of conditional overexpression HPV16 E6 animal model at ROSA26 site

[0094] 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:

[0095] (1) sgRNA design and evaluation of off-target effects

[0096] sgRNA design for conditional overexpression of HPV16 E6 at the ROSA26 locus in C57BL / 6 mice, and evaluation of off-target effects of candidate sgRNAs, such as figure 2 shown. Screening to get sgRNA(matching forward strandofgene):

[0097] CUCCAGUCUUUCUAGAAGAUGGG.

[0098] (2) co-incubating the obtained sgRNA with the Cas9 protein to prepare a Cas9 / sgRNA mixture;

[0099] (3) Gene editing strategy and targeting vector construction

[0100] The overall strategy of gene editing is as follows: f...

specific Embodiment 2

[0136] Specific embodiment 2: Carry out the construction of conditional overexpression HPV16 E7 animal model at H11 site

[0137] 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:

[0138] (1) sgRNA design and evaluation of off-target effects

[0139] sgRNA design for conditional overexpression of HPV16 E7 at the H11 site of C57BL / 6 mice, and evaluation of off-target effects of candidate sgRNAs, such as Figure 14 . Screening to get sgRNA(matching forward strand ofgene):

[0140] GAACACUAGUGCACUUAUCCUGG.

[0141] (2) co-incubating the obtained sgRNA with the Cas9 protein to prepare a Cas9 / sgRNA mixture;

[0142] (3) Gene editing strategy and targeting vector construction

[0143] The overall strategy of gene editing is as follows: Figure 13...

specific Embodiment 3

[0177] Specific Example 3: Generation of conditional site-directed HPV16 E6 / E7 double knock-in mice

[0178] Method for obtaining conditional site-directed HPV16 E6 / E7 double knock-in mice by hybridization: the ROSA26 site conditionally overexpressing HPV16 E6 homozygous mice constructed in Example 1 and the H11 site conditionally overexpressing HPV16 E7 in Example 2 The homozygous mouse model was crossed to obtain the positive offspring of conditional site-directed HPV16 E6 / E7 double knock-in mice. The offspring after heterozygosity are 100% homozygous, and genotyping may not be performed.

[0179] The present invention provides a conditional site-specific HPV E6 / E7 double transgenic mouse model established by transgenic technology, so as to obtain an animal model that can be genetically stable and conforms to the true pathological state of HPV infection. This model will be used to study the in vivo function analysis of the key HPV oncogene E6 / E7, the exploration of disease ...

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Abstract

The invention provides a construction method of a mouse model realizing conditional site-specific double-overexpression of HPV E6/E7 genes. The construction method comprises the following steps: 1, acquiring mice: (1) respectively designing and acquiring sgRNAs; (2) respectively preparing Cas9/sgRNA mixtures; (3) constructing a targeting vector; (4) carrying out microinjection and acquiring F0-generation mice, including F0-generation mice with HPV E6 knocked in at the specific ROSA26 site and F0-generation mice with HPVE7 knocked in at the specific H11 site; (5) acquiring F1-generation mice; and (6) acquiring F2-generation positive homozygote mice; and 2, acquiring conditional site-specific HPV E6/E7 double-knock-in model mice: hybridizing the two F2-generation positive homozygote mice toobtain the conditional site-specific HPV E6/E7 double-knock-in model mice. According to the method provided by the invention, the animal model which can be stably inherited and accurately regulated and controlled can be obtained.

Description

technical field [0001] The invention relates to a method for constructing a conditional site-specific double overexpression HPV E6 / E7 gene mouse model, belonging to the field of biotechnology. Background technique [0002] Cervical cancer is one of the most common malignant tumors in women. Its incidence rate is second only to breast cancer in gynecological malignancies. It has a complex pathogenesis process, a high incidence rate, and a trend of younger patients, which seriously threatens women's lives and health. Its occurrence and development is a complex and multi-step process, which is the result of long-term synergistic effects of various carcinogenic factors, including human papillomavirus (HPV) infection, gene mutation, and immune escape. A large number of epidemiological and molecular biology studies have confirmed that HPV is closely related to reproductive tract epithelial malignant tumors and precancerous lesions, and is the main etiological factor of cervical ca...

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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