Construction method of STAT3 mitochondrial localization conditional gene knock-in mouse model

A technology of gene knock-in and positioning conditions, applied in the field of bioengineering, can solve problems such as functions and molecular mechanisms that have not been fully elucidated

Pending Publication Date: 2020-08-07
XUZHOU MEDICAL UNIV
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Problems solved by technology

However, the function and molecular mechanism of STAT3 after mitochondrial localization have not been fully elucidated

Method used

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  • Construction method of STAT3 mitochondrial localization conditional gene knock-in mouse model
  • Construction method of STAT3 mitochondrial localization conditional gene knock-in mouse model
  • Construction method of STAT3 mitochondrial localization conditional gene knock-in mouse model

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Embodiment

[0047] This embodiment provides a method for constructing a STAT3 mitochondrial localization conditional gene knock-in mouse model, such as figure 1 As shown, it specifically includes the following steps:

[0048] (1) Construction of a recombinant targeting vector for conditional gene knock-in of STAT3 mitochondrial localization: Synthesize the Cox4singal-STAT3 gene, and then insert the Cox4singal-STAT3 gene at the Rosa26 gene locus into the CAG-LSL- Cox4 singal-STAT3-WPRE-pA expression cassette, and then obtain the recombinant targeting vector of conditional gene knock-in of the STAT3 mitochondrial localization; the recombinant targeting vector contains 3.3kb 5' homology arm, CAG-LSL-Cox4singal-STAT3 - WPRE-pA and 3.3 kb 3' homology arm.

[0049] (2) The recombinant targeting vector, Cas9 mRNA and gRNA with the sequence 5'-GGGGACACACTAAGGGAGCTTGG-3' were transfected together into fertilized eggs of C57BL / 6J donor mice.

[0050] (3) Transplant the fertilized egg into the ute...

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Abstract

The invention relates to a construction method of an STAT3 mitochondrial localization conditional gene knock-in mouse model. The method comprises the following steps: firstly, constructing an STAT3 mitochondrial localization conditional gene knock-in recombinant targeting vector, transfecting the recombinant targeting vector to fertilized ova of a donor mouse, and then transplanting the donor mouse fertilized ova into the uterus of a fake pregnant mother mouse to produce a chimeric mouse; mating the chimeric mouse with a C57BL/6J mouse to obtain an F1 generation; mating the F1 generation witha mouse of tissue-specific expression Cre recombinase to obtain a double-hybrid mouse; and are intercrossing the double-hybrid mouse and an STAT3<f/+> or STAT3<f/f> mouse, and inducing the obtained offspring by tamoxifen to obtain the STAT3 mitochondrial localization conditional gene knock-in mouse model. According to the mouse model disclosed by the invention, the STAT3 gene is expressed in specific tissues and cell types, and an ideal model is provided for researching the action mechanism of STAT3 in specific cell development and differentiation, body metabolism and tumor occurrence and development.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for constructing a STAT3 mitochondrial localization conditional gene knock-in mouse model. Background technique [0002] Gene knock-in refers to designing experiments at the molecular level for a gene whose structure is known but whose function has yet to be elucidated, overexpressing the gene in vivo, and then observing the experimental animals as a whole to infer the function of the corresponding gene. [0003] Signal transducer and activator of transcription 3 (STAT3) is a member of a protein family that plays a role in signal transduction and transcription factor regulation. regulation of normal cells. More and more studies have shown that abnormal activation of STAT3 is closely related to a variety of human malignant tumors. Inhibiting the overexpression of STAT3 is one of the means to treat tumors. In addition, STAT3 mediated both the expressi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90A01K67/027
CPCA01K67/0278A01K2217/07A01K2227/105A01K2267/0331C12N15/907
Inventor 杨晶陈欣
Owner XUZHOU MEDICAL UNIV
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