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Construction method and application of conditional Yap1 gene knock-in mouse

A construction method and conditional technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as unfavorable experimental research, lack of animal models of oncogenes, and lack of specificity for lung squamous cell carcinoma

Active Publication Date: 2020-11-10
THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The existing mouse models of lung squamous cell carcinoma have many obvious defects: (1) spontaneous tumor formation is abnormally slow, often taking several months or even a year, which is very unfavorable for experimental research; (2) the pathological types of spontaneous tumor tissue are not pure, the same There are multiple pathological subtypes in tumor tissue; (3) Since the knockout of tumor suppressor genes does not have the specificity of lung squamous cell carcinoma, there is a lack of research on animal models that introduce tumor-promoting genes

Method used

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  • Construction method and application of conditional Yap1 gene knock-in mouse
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  • Construction method and application of conditional Yap1 gene knock-in mouse

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preparation example Construction

[0074] The preparation method of the linearized plasmid is not particularly limited in the present invention, preferably including: transforming Escherichia coli with a carrier plasmid; extracting the plasmid by shaking bacteria; purifying the plasmid; verifying the plasmid by enzyme digestion; linearizing the plasmid by enzyme digestion; verifying linearization by enzyme digestion Plasmid; linearize the plasmid by electrophoresis, cut the gel to recover the target fragment; verify the recovered linearized product by enzyme digestion, and obtain the linearized vector plasmid. In the present invention, there are no special limitations on the experimental methods and reagents involved in the above-mentioned preparation method of the linearized plastid, and conventional methods and reagents in the art can be used.

[0075] The method of the present invention is not particularly limited to the ES targeting method, and conventional methods in the field can be used, preferably includ...

Embodiment 1

[0104] Construction of targeting vector:

[0105] (1) Use KpnI / PmII to digest the basic skeleton containing the ROSA26 gene: ROSA26-5'arm-rBGpA(r)-MSC-SDA-sdNeo cassette-SDA-ROSA26-3'arm;

[0106] (2) Directly synthesize the plasmid human SP-C promoter-mutant mouse Yap1 CDS, clone it into the pUC57 vector, use EcoRV to digest the pUC57-Human SP-C promoter-mutant mouse Yap1 CDS vector, and recover the 5252bp band, namely For Human SP-C promoter-mutant mouse Yap1 CDS;

[0107] (3) The target fragment: Human SP-C promoter-mutant mouse Yap1 CDS was ligated into the enzyme-cut backbone using In-fusion ligase to obtain the SP-C promoter-Yap1-ROSA26-KI431 targeting vector .

Embodiment 2

[0109] Determination of targeted clones: Delivery of linearized vectors to ES cells (C57BL / 6) by electroporation, followed by G418 drug selection, selection Figure 5 The indicated regions were screened by PCR, and a total of 91 drug-resistant clones were obtained, and the samples labeled 1F2, 1D4, 1B5, 1G6, 1H5, 1D8 and 1D9 were identified as potential target ES clones, and the results were as follows Figure 6 As shown; select the same region, perform SouthernBlot on 1B5, 1D4, 1D8, 1F2, 1G6 and 1H5 samples, and the predicted fragment sizes are:

[0110] Neo Probe (containing 5’HA)–9.7Kbp–Hind III;

[0111] Neo Probe(containing 3’HA)–12.7Kbp–Kpn I; the results are as follows Figure 7 As shown, all six ES clones (1B5, 1D4, 1D8, 1F2, 1G6 and 1H5) were confirmed to be correct.

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Abstract

The invention provides a construction method and application of a conditional Yap1 gene knock-in mouse, and relates to the technical field of gene targeting. The nucleotide sequence of a conditional Yap1 knock-in targeting vector is shown as SEQ ID NO.1, and a mouse (Yap1KI) knocked in by the conditional Yap1 knock-in targeting vector can be constructed directly by utilizing the conditional Yap1 knock-in targeting vector. The Yap1 gene knock-in mouse is constructed for the first time, and can be used for constructing a lung cancer model, and an animal model of the Yap1 gene knock-in mouse hasthe characteristic of rapid tumor formation.

Description

technical field [0001] The invention belongs to the technical field of gene targeting, and in particular relates to a construction method and application of a conditional Yap1 gene knock-in mouse. Background technique [0002] Lung cancer is currently the largest malignant tumor in human beings, and its morbidity and mortality both occupy the first place. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer patients; 60-80% of NSCLC is lung adenocarcinoma, and 20-40% is lung squamous cell carcinoma. Tumor-initiating cells (TICs) are a group of stem cells with the ability of "self-renewal" and "multi-lineage differentiation". important reason. [0003] The existing mouse models of lung squamous cell carcinoma have many obvious defects: (1) spontaneous tumor formation is abnormally slow, often taking several months or even a year, which is very unfavorable for experimental research; (2) the pathological types of spontaneous tumor tissue are not pure, the sa...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/12A01K67/02
CPCA01K67/0278A01K2217/072A01K2227/105A01K2267/0331C07K14/47C12N15/8509
Inventor 孙建国李奉孟令鑫廖星芸赵先兰余永新廖荣霞
Owner THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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