Construction method and application of spontaneous gastric precancerous lesion animal model
A technology of gastric precancerous lesions and construction methods, applied in the field of animal model construction of spontaneous gastric precancerous lesions, can solve the problems of time-consuming, large individual differences, and low clinical fit
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Embodiment 1
[0045] Animal model ATP4a + / - Mouse construction.
[0046] 1.1 Gene editing
[0047] Retrieve the C57BL / 6J mouse gene database MGI (http: / / www.informatics.jax.org / ), and retrieve the wild-type gastric H + -K + - ATPase 4a gene (ATP4a, ATPase, gastric H + / K + exchanging alphapolypeptide), MGI gene number: 88113, NCBI Gene number: 11944, Ensembl: ATP4a (ENSMUSG00000005553). Located in mouse chromosome 7: NC_000073.7, gene start and stop sequence: 30411634~30424959. Its gene structure is as follows figure 1 shown.
[0048] The gene transcript ATP4a-201 (Ensembl number: ENSMUST00000005692.13) has 22 exons, 50 structural domains and features. CRISPR / Cas9 technology knocks out exons exon5-11 (introns between consecutive exons 5-11) in the ATP4a gene transcript of C57BL / 6J mice, such as figure 2 shown.
[0049] After cleavage, connect the cleavage sites 1 and 2 to form the ATP4a silent expression gene after gene knockout, see image 3 . Cas9 mRNA and gRNA were obtained ...
Embodiment 2
[0066] 1. Materials and methods
[0067] 1.1 Animals
[0068] 4-5 weeks healthy SPF grade littermate first generation (F1) wild-type ATP4a + / + and ATP4a + / - Mice, weighing 20-25 g, provided by Shanghai South Model Organism Co., Ltd., experimental animal license number: SCXK 2014-0002, animal qualification certificate number: 312024300003479, self-cultivated to the F3 generation for future use; animal breeding environment: Guangzhou Traditional Chinese Medicine University Animal Experiment Center SPF Animal Laboratory, environmental certification number: SYXK (Guangdong) 2013-0001.
[0069] 1.2 Drugs and reagents
[0070] Anti-rabbit primary antibody MUC2 (lot number: Ab76774), anti-rabbit Ki-67 (lot number: Ab16667), product of abcam USA; AMACR (lot number: NBP1-87168), product of Novous company; anti-mouse p53 (lot number: CBL404), Products of Millipore Company; Immunohistochemistry-related secondary antibodies: Mouse / Rabbit Universal Immunohistochemistry Detection Kit (G...
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