Construction method of humanized CYP2D6*10 transgenic mouse model

A technology of transgenic mice and mice, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve problems such as differences in the types of metabolic enzymes

Pending Publication Date: 2021-04-09
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for constructing a human CYP2D6*10 transgenic mouse model to solve the existing problem that the types of metabolic enzymes in mice used for in vivo research are different from those in humans

Method used

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  • Construction method of humanized CYP2D6*10 transgenic mouse model
  • Construction method of humanized CYP2D6*10 transgenic mouse model
  • Construction method of humanized CYP2D6*10 transgenic mouse model

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Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1 The method involved in the present invention belongs to in situ knock-in of humanized CYP2D6*10 gene

[0021] like figure 1Shown, the present invention first constructs ES cell targeting plasmid, comprising 3.0kb 5 ' homology arm, hCYP2D6promoter-hCYP2D6 genomic DNA-polyA, PGK-Neo-polyA, 3.0kb 3 ' homology arm and MC1-TK-polyA negative screening mark. After the targeting plasmid was linearized, ES cell targeting was performed, and the targeting vector was homologously recombined with the C57BL / 6J mouse ES cell genome to obtain genetically recombined ES cells with the human CYP2D6 gene inserted between the 4th and 5th exons, and through G418 pressure Screen to obtain resistant ES cells. Positive ES cell clones with correct homologous recombination in both arms were screened by PCR and sequencing. After obtaining positive clones, the Cas9-gRNA vector targeting the target gene was transfected into positive ES cell clones with correct homologous recombinatio...

Embodiment 2

[0022] Embodiment 2 utilizes restriction endonuclease or gene sequencing to verify the targeting plasmid

[0023] like figure 2 As shown in the figure, a targeting plasmid was constructed, and the target sequence was verified by restriction endonuclease and gene sequencing to ensure the correctness of the sequence. Configure the enzyme digestion reaction system as shown in the following table:

[0024] React Component Volume(μl) ddH2O 15.9 10×Buffer 2 EcoRI 1.5 Plasmid DNA 0.6 Total 20

[0025] Mix the system and put it into a 37°C water bath. After 2 hours of reaction, add an appropriate amount of Gel loading buffer and mix it up to perform agarose gel electrophoresis. After the bands are separated, take pictures. And 260bp (the small fragment may not be seen due to the low content) and other 5 bands. The correct plasmid was verified by enzyme digestion and sent to a sequencing company for sequencing.

Embodiment 3

[0026] Example 3 Screening of obtained ES cell clones by PCR technology

[0027] like image 3 As shown, the PCR identification scheme of homologous recombination positive clones: after the genomic DNA of resistant ES cell clones is extracted, the homologous recombination positive clones are screened by long-segment PCR. The primer positions are as follows image 3 shown. The 5' arm homologous recombination positive clone should amplify a 3.6kb fragment, and the negative clone should have no product; the 3' arm homologous recombination positive clone should amplify a 6.0kb fragment, and the negative clone should have no product. KO positive clones should amplify a 2.2kb fragment, and negative clones should have no product.

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Abstract

The invention discloses a construction method of a humanized CYP2D6*10 transgenic mouse. According to the method, the homologous recombination principle is utilized, an ES targeting mode is adopted, an expression cassette of hCYP2D6*10 is knocked into an exon4-5 site of the Cyp2d22 gene in a fixed-point mode, and then a Cyp2d gene cluster is knocked out in a Crispr / Cas9 mode; firstly, a targeting vector is prepared, and ES cells are electrically transfected after the vector is linearized; through long-fragment PCR identification, positive clones of correct homologous recombination are obtained; and the positive ES cell clone is amplified to be injected into a blastocyst of a C57BL / 6J mouse to obtain a chimeric mouse. Compared with a non-transgenic mouse, the humanized CYP2D6*10 transgenic mouse bred by the invention can express a human CYP2D6*10 type gene (dominant proportion allele of Chinese population), and can replace human beings to be used for developing pharmacokinetic and pharmacodynamic related research of substrate drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for constructing a CYP2D6*10 transgenic mouse model, in particular to a method for expressing the main genotype CYP2D6*10 in the Chinese CYP2D6 family in the transgenic mouse. Background technique [0002] CYP2D6 is one of the most genetically polymorphic subtypes in the CYP450 family and mediates in vivo elimination of at least 20% of clinically administered drugs (Gaedigk A, Sangkuhl K, Whirl-Carrillo M, Klein T, Leeder JS. Prediction of CYP2D6 phenotype from genotype across world populations. Genet Med. 2017;19(1):69-76;Zhou SF,Liu JP,Lai XS.Substrate specificity,inhibitors and regulation of human cytochrome P450 2D6 and implications in drugdevelopment.Curr Med Chem.2009;16 (21):2661-805.). Up to now, 141 CYP2D6 alleles (excluding non-amino acid coding region variation) have been reported, and the protein functions encoded by them have extensive enzymatic activity changes,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027C12N15/53
CPCC12N15/8509C12N9/0071A01K67/0278C12Y114/14001C12N2800/107A01K2217/072A01K2227/105A01K2267/03
Inventor 陈冰冰钱建畅胡国新李军伟肖健林丽
Owner WENZHOU MEDICAL UNIV
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