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Animal model with male reproductive disorders, as well as preparation method and application thereof

A fertility disorder and animal model technology, applied in biochemical equipment and methods, biological testing, microbiological determination/inspection, etc., can solve the problems of male mouse fertility decline, sperm-egg combination ability decline, and inability to complete the fertilization process, etc.

Inactive Publication Date: 2013-10-23
上海南方模式生物研究中心 +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0011] Existing evidence has shown that serine proteases play an important role in the fertilization process and participate in physiological processes such as sperm capacitation and sperm-egg integration. For example, PCSK4 is a member of serine protease subtilisin-like, which is highly expressed in male germ cells. Genetic mice have normal spermatogenesis and development, but the sperm-egg binding ability is reduced, and the fertilization process cannot be completed. It is speculated that it may participate in some precursor proteins synthesized by male germ cells, such as proenkephalin, pronerve growth factor, propituitary adenylate cyclase-activating peptide (proPACAP), the enzymatic processing of IGF-1 receptor, hepatocyte growth factor receptor and ADAM family proteins, the related mechanism needs to be further studied[18]; Prss21 and Acrosin are the other two serines expressed by sperm Protease, in vitro studies have found that it is involved in the combination of sperm and eggs, but single gene knockout does not affect male reproduction [19], only when the double gene knockout male fertility will decline [20]

Method used

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  • Animal model with male reproductive disorders, as well as preparation method and application thereof
  • Animal model with male reproductive disorders, as well as preparation method and application thereof
  • Animal model with male reproductive disorders, as well as preparation method and application thereof

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Experimental program
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preparation example Construction

[0109] Preparation of Prss37 knockout mice

[0110] The targeting vector was linearized with NotI and electroporated into SCR012ES cell line. Resistant cells were selected with G418 and ganciclovir. DNA was extracted from 128 clones, and positive clones with homologous recombination with the targeting vector were identified by PCR. The identification primers for the 5' and 3' homology arms were P1 (5'-CGAGCATCCTGGCTTATC-3', SEQ ID NO: 23), P2 (5'-CCACTCCCACTGTCCTTT-3', SEQ ID NO: 24) and P3 (5'- TTTATTAGGAAAGGACAGTGGGAGT-3', SEQ ID NO: 25), P4 (5'-TCTGTGAAGTAGGGATGGGTTGT-3', SEQ ID NO: 26), the PCR products are 5520bp and 4459bp, respectively. C57BL / 6J blastocysts were injected with two independent ES cell lines and subsequently transferred into pseudopregnant female mice to obtain chimeric offspring. The chimeric males were mated with C57BL / 6J females to produce heterozygous mice. All mice were housed in temperature-controlled cages, and the room was kept on a 12h day / 12h...

Embodiment 1

[0137] Tissue expression patterns and cell type-specific localization patterns of the Prss37 gene

[0138] cDNA samples were prepared by extracting RNA from 16 mouse tissues, including brain, cerebellum, thymus, heart, lung, liver, spleen, kidney, stomach, small intestine, bladder, skeletal muscle, testis, epididymis, ovary and uterus. Using specific primers for reverse transcription PCR and real-time PCR detection, it was found that a unique Prss37 transcript was specifically and highly expressed in testis tissue ( figure 1 ). By studying the testis and epididymis tissues of male mice at 7, 14, 18, 21, 23, 28, and 42 days after birth, it was further found that Prss37 gene transcription began in the testes of male mice at 23 days after birth, and began to be detected at 28 days after birth to the Prss37 protein. Neither Prss37 mRNA nor Prss37 protein was found in epididymis tissue, indicating that Prss37 protein was hardly present in spermatozoa in epididymis ( figure 2 )....

Embodiment 2

[0140] Preparation and Identification of Prss37 Gene Knockout Mouse Model

[0141] gene targeting strategies such as Figure 4 As shown in A, we deleted exon3 and exon4 of Prss37 gene by ET clone and ES cell homologous recombination, and replaced the deleted part with Neo resistance gene, in which the 5' homology arm was 4768bp long and the 3' homology arm was 3543bp long. Eight positive ES cell clones were identified by PCR method ( Figure 4 B). Two of these independent ES cell clones were used for blastocyst injection to obtain chimeric mice. Male chimeras were mated with C57BL / 6J female mice to obtain heterozygotes with mixed genetic background (129-C57). Three genotype mice conforming to Mendelian inheritance rules were obtained by mating heterozygous male mice and heterozygous female mice. And strictly from the DNA ( Figure 4 C), RNA ( Figure 4 D) and protein ( Figure 4 E-F) Levels for genotype verification.

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Abstract

The invention provides an animal model with male reproductive disorders of a non-human mammalian animal, as well as a preparation method and application of the animal model, and further identifies the functions of a Prss37 gene and a protein. The Prss37 gene of the animal model is inactivated, thereby affecting the appearance of ADAM3 in mature sperms produced by the animal model, indirectly affecting the combination of the sperms-zona pellucida and the migration of the sperms from the uterus to the oviduct and resulting in the serious male reproductive disorders. The model, the Prss37 gene and the protein thereof can be used for screening ADAM3 ripening accelerators, as well as sterility infertility treatment agents or contraceptive medicaments.

Description

technical field [0001] The present invention relates to the field of biotechnology. More specifically, the present invention relates to a method for establishing an animal model of male fertility disorder in a non-human mammal and its use. Background technique [0002] Human reproduction is related to the quality of the population, and in modern life, due to work pressure and various environmental factors, people's reproductive quality is declining year by year. Therefore, research on human reproduction has become a "hot spot" for scientific researchers . [0003] Male fertility is a complex process that is affected by many different factors, such as normal sperm production, good semen quality, adequate sperm count, normal sperm morphology and motility, etc. [1]. According to reports, in Western countries, 1 / 6 couples have difficulty in having children. Half of them were due to male factor infertility (MFI). One-third of MFIs are associated with chromosomal abnormalities...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12Q1/68G01N33/573G01N33/68A01K67/027A61K49/00A61K45/00A61P15/00A61P15/16
Inventor 王铸钢沈春玲刘建兵匡颖费俭
Owner 上海南方模式生物研究中心
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