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Building method of spontaneous squamous cell lung carcinoma mouse models

A technology for constructing methods and mouse models, which can be applied to other methods of inserting foreign genetic materials, chemical instruments and methods, and botanical equipment and methods, etc. Experimental research, etc.

Active Publication Date: 2020-11-24
THE SECOND AFFILIATED HOSPITAL ARMY MEDICAL UNIV
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Problems solved by technology

However, there are currently existing mouse models of lung squamous cell carcinoma, which have many obvious defects, mainly: (1) spontaneous tumor formation is extremely slow, often taking several months or even a year, which is very unfavorable for experimental research; (2) pathological changes in spontaneous tumor tissue The type is not pure, and there are multiple pathological subtypes in the same tumor tissue; (3) Since the knockout of tumor suppressor genes does not have the specificity of lung squamous cell carcinoma, etc., there is a lack of research on animal models that introduce oncogenes

Method used

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  • Building method of spontaneous squamous cell lung carcinoma mouse models
  • Building method of spontaneous squamous cell lung carcinoma mouse models
  • Building method of spontaneous squamous cell lung carcinoma mouse models

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Embodiment 1

[0054] 1. Design and construction of conditional Yap1 knock-in targeting vector: A mouse Yap1 conditional knock-in model was established in C57BL / 6 mice. (See figure 1 and figure 2 )

[0055] 1. Vector construction:

[0056] (1) Order BAC, and prepare the primers required for vector construction; (2) Extract BAC by shaking the bacteria, and prepare the basic vector plasmid (KI431-basic vector); (3) PCR amplify the target fragment Yap1 from BAC DNA, and detect the amplification by electrophoresis. Amplify the product, cut the gel to recover the target fragment; (4) connect the target fragment to the basic vector (cut the basic vector with pmlI and KpnI); (5) transform the ligated product into a competent state, spread it on a plate and culture it overnight; (6) pick a single colony , positive clones were screened by colony PCR; (7) small plasmids were extracted from colony PCR positive clones, and identified by enzyme digestion; (8) positive clones identified by enzyme dige...

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Abstract

The invention provides a building method of spontaneous squamous cell lung carcinoma mouse models, and relates to the technical field of building of mouse models. According to the building method of the spontaneous squamous cell lung carcinoma mouse models, by designing targeting vectors with conditional Yap1 knocked in, mice (Yap1KI) with the conditional Yap1 knocked in are built, due to tumor-promoting action of Yap1, a spontaneous squamous cell lung carcinoma has the character of fast tumor formation, then according to standard design of transgenic mice, negative selection markers (DTA) andpositive selection markers (Neo) are also introduced into the targeting vectors, thus positive clones can be conveniently and fast obtained, and by conducting hybridization on mice with Trp53 genes knocked out and the Yap1KI mice, Yap1KI / Trp53KO mice are obtained at last, so that building of the spontaneous squamous cell lung carcinoma mouse models is completed.

Description

technical field [0001] The invention belongs to the technical field of mouse model construction, and in particular relates to a method for constructing a spontaneous lung squamous cell carcinoma mouse model. 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, 20-40% is lung squamous cell carcinoma, and for lung cancer, especially lung squamous cell carcinoma Animal models are essential for cancer treatment and mechanism research. However, there are currently existing mouse models of lung squamous cell carcinoma, which have many obvious defects, mainly: (1) spontaneous tumor formation is extremely slow, often taking several months or even a year, which is very unfavorable for experimental research; (2) pathological changes in spontaneous tumor tissu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/12A01K67/027
CPCC12N15/8509C12N15/907C07K14/82A01K67/0278C12N2800/107A01K2207/15A01K2217/072A01K2227/105A01K2267/03
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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