Gene related to animal infertility, sgRNA, application and method for constructing animal model

An animal model and gene technology, applied in the field of animal models, can solve the problems of large differences between individuals and low success rate of induction of female infertility animal models.

Active Publication Date: 2021-08-03
YUNNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In view of this, the purpose of the present invention is to provide a gene related to animal infertility, sgRNA and a method for applying and constructing an animal model, which can solve the problem of low success rate of induction of female infertility animal models and large differences among individuals induced by the same batch. technical issues

Method used

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  • Gene related to animal infertility, sgRNA, application and method for constructing animal model
  • Gene related to animal infertility, sgRNA, application and method for constructing animal model
  • Gene related to animal infertility, sgRNA, application and method for constructing animal model

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Experimental program
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Effect test

Embodiment 1

[0049] according to figure 1 The process shown in the construction of dguok deletion (Dguok(- / -)) mice:

[0050] Entrusted Qingke Kunming Synthesis Department to design dguok sgRNA (SEQ ID NO.3-6).

[0051] Microinject dguok sgRNA and CAS9 mRNA synthesized in vitro into the nuclear region of fertilized eggs of C57BL / 6 strain mice (Nanjing Model Animal Center); and then transplant the fertilized mouse oocytes into the oviducts of pseudopregnant C57BL / 6 strain mice , get F 0 Generate mice and carry out genotype identification (using primers M-DguoK-F, M-DguoK-R and M-Dguok-deletion);

[0052] will positive F 0 F 1 Generation mice, using the above method for genotype identification, the positive F 1 Heterozygous mice (M-DguoK-F and M-DguoK-R produce a 1.7kb band, and M-DguoK-R and M-Dguok-deletion produce a band of about 800bp) self-crossed to produce wild (only M-DguoK-F and M-DguoK-R produce a 1.7kb band), heterozygous and homozygous F 2 Generation mice (only M-DguoK-R a...

Embodiment 2

[0055] The mouse toes were clipped and numbered, and the toes of the upper limbs were from left to right, representing ten digits. The toes of the lower limbs run from left to right, representing single digits. Put the clipped toe into the lysate and extract the DNA. Utilize the primer combination of M-DguoK-F and M-DguoK-R and M-DguoK-R and M-Dguok-deletion to carry out PCR, the result is as follows image 3 Middle A and image 3 As shown in middle B, dguok(- / +) heterozygous mice (produce two bands of about 1.7kb and 1kb), wt wild mice (produce only one band of 1.7kb), dguok- / - homozygous small Mouse (produces only one 1 kb band).

Embodiment 3

[0057] The genotyped wild mice and homozygous mice were sacrificed and dissected, and their liver and lung tissues were taken. The protein was extracted after the tissue was crushed, and the expression of Dguok at the protein level was detected by Western Blot. There was no expression of Dguok in the tissues of homozygous mice. The result is as image 3 Shown in C.

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Abstract

The invention provides a gene related to animal infertility, sgRNA, application and a method for constructing an animal model, and relates to the technical field of animal models. The invention provides a gene related to animal infertility. The gene comprises an animal dguok gene. The sgRNA is designed on the basis of the gene, a female infertility animal model is constructed, the model is 100% infertility, and the expression is that ova cannot be mature, cannot be successfully fertilized, cannot be split and cannot enter an 8-cell stage and a blastocyst stage; and the female infertility mouse model is helpful for deeply researching a specific molecular mechanism of female infertility on a molecular level, and is helpful for researching and developing related medicines for treating infertility.

Description

technical field [0001] The invention belongs to the technical field of animal models, and specifically relates to a gene related to animal infertility, sgRNA and its application and a method for constructing an animal model. Background technique [0002] At present, infertility has become a common and frequently-occurring disease. In the field of reproductive medicine, the preparation of animal models of infertility is the basis for carrying out related research. At present, there are many factors leading to infertility, such as ovulation disorders (central nervous system anovulation, hypothalamic anovulation, pituitary anovulation, ovarian anovulation, polycystic ovary syndrome, etc.), fallopian tube factors (inflammation or obstruction), embryonic factors (fertilization abnormalities, cleavage abnormalities, early embryo developmental block, etc.), uterine factors (developmental deformities, fibroids and endometrial lesions, etc.), in addition to pelvic factors, cervical f...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/113C12N15/89C12N15/877A01K67/027
CPCC12N9/1229C12N15/1137C12N15/89C12N15/8775A01K67/0276C12Y207/04008C12N2310/20A01K2207/15A01K2217/075A01K2227/105A01K2267/03
Inventor 孙建伟桑磊高亚可董瑞沈俊岭陈慧诚
Owner YUNNAN UNIV
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