Construction method of mouse model simulating human acute pancreatitis

A technology of acute pancreatitis and mouse models, which is applied in the fields of botany equipment and methods, biochemical equipment and methods, enzymes, etc., and can solve problems such as doubting the credibility of experimental results

Active Publication Date: 2017-10-24
NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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  • Claims
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Problems solved by technology

The conclusions of the above two experiments are partly contradictory, and the reason is that the transgenic mice used are defective: the promoters used to express the target gene are all rat-derived elastase, not human-derived elastase, so from There are fundamental doubts about the credibility of the experimental results

Method used

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  • Construction method of mouse model simulating human acute pancreatitis
  • Construction method of mouse model simulating human acute pancreatitis
  • Construction method of mouse model simulating human acute pancreatitis

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Embodiment 1

[0038] Example 1 Construction of targeting vector plasmid containing PRSS1 gene

[0039] 1. The construction of the carrier: the schematic diagram of the construction is as follows figure 1 shown.

[0040] 1. Construct an intermediate vector and construct the target fragment into the pStart-K backbone;

[0041] 2. Amplify the fragment to be recombined;

[0042] 3. Perform homologous recombination of the amplified fragment with BAC;

[0043] 4. Screen the correct clone;

[0044] 5. Amplify the target fragment on the BAC and send it for testing;

[0045] 6. Perform a loop test on the correctly sequenced BAC.

[0046] 7. Verify the correctness of the obtained carrier.

[0047] 8. Transform Escherichia coli with the vector plasmid (that is, the targeting vector plasmid containing the PRSS1 gene, whose nucleotide sequence is shown in SEQ ID NO.1), to obtain the E. coli liquid transformed with the vector plasmid.

[0048] 2. Plasmid preparation

[0049] 1. Streak the bacteri...

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Abstract

The invention discloses a construction method of a mouse model simulating human acute pancreatitis. Targeting vector plasmids containing PRSS1 genes are constructed and transferred to mouse embryonic stem cells electrically, and the mouse embryonic cells carrying the recombinant PRSS1 genes are obtained; the mouse embryonic cells carrying the recombinant PRSS1 genes are micro-injected into albinistic mouse blastodermal cells, the blastodermal cells receiving injection are transplanted to the uteri of pseudo-pregnant maternal mice, and chimeric mice are obtained after transplanting; male chimeric mice with the chimerism higher than 25% and wild C57BL / 6J female mice are mated, the next generation of mice are bred, and mice carrying PRSS1 genes are screened. The phenotype of the model mouse is greatly identical to the pathogenetic process of human acute pancreatitis, and the specific animal model completely simulating human acute pancreatitis from gene to phenotype is obtained and can be further widely applied to researches on pathopoiesis, pathomechanism and the like of human PRSS1 as well as screening and gene therapy tests of medicines for treating pancreatitis and the like.

Description

Technical field: [0001] The invention belongs to the field of animal model construction, in particular to a method for constructing a mouse model of human acute pancreatitis. Background technique: [0002] Acute pancreatitis (AP) is a clinically common and potentially fatal acute abdomen. Although great progress has been made in the diagnosis and treatment of AP, the fatality rate is still about 10%, and the severe cases reach 50%. The most important reason is that the specific mechanism of AP initialization has not been fully understood, and there is no way to guide the development of new and more effective drugs for the treatment of pancreatitis. Over the past 20 years, a large number of studies based on animal models of experimental pancreatitis have found that premature activation of trypsinogen in acinar cells plays a key role in the initialization of AP. However, the specific mechanism of trypsingen activation and its role in the development of pancreatitis have not ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/877A01K67/027
CPCA01K67/0278A01K2227/105A01K2267/0368C12N9/6427C12N15/8509C12N15/8775C12Y304/21004
Inventor 张国伟周杰阚和平钱建平林振宇任昰婧范宇
Owner NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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