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CN-121986757-A - Gene editing mouse model construction method for researching calcium library function and low fertility in mature sperm and application thereof

CN121986757ACN 121986757 ACN121986757 ACN 121986757ACN-121986757-A

Abstract

The invention relates to the technical field of animal model construction, in particular to a method for constructing a gene editing mouse model for researching calcium library function and low fertility in mature sperms and application thereof, wherein the model utilizes tamoxifen to induce and Ddx ERT2 promoter to drive Cre recombinase to express, and three subtypes of IP3R are knocked out in sperms specifically and simultaneously so as to eliminate compensatory effect among the subtypes, and the method is used for researching the action and mechanism of the gene editing mouse model in male fertility. The model mice are a good model for evaluating sperm dysfunction after two rounds of induced knockout, the fertility is seriously impaired, the number of sperms and the movement level are reduced, and the acrosome response is abnormal, so that the model mice are used for screening and improving the treatment drugs for male sterility diseases caused by sperm dysfunction, especially the acrosome response in advance in future. In addition, by screening optimization of IP3R agonists and calcium signaling platforms, it was further confirmed that this mouse model was a good model for studying sperm calcium pool-mediated calcium signaling.

Inventors

  • ZHANG XIAONING
  • ZENG XUHUI
  • CHEN CHEN
  • Wang Louxing
  • CAI ZIYI
  • YANG FENGYU

Assignees

  • 南通大学

Dates

Publication Date
20260508
Application Date
20260204

Claims (8)

  1. 1. The method for constructing the gene editing mouse model for researching the calcium library function and the low fertility in mature sperms is characterized by comprising the following steps: Step 1, mating a Loxp mouse homozygous for the three sites of the IP3R with a constructed B6-Igs2 (Ddx4CreERT2) NTUXi mouse to generate a three-site heterozygous Loxp mouse with Ddx4CreERT2 ; Step 2, further mating with an IP3R three-locus homozygous Loxp mouse to obtain a target mouse for conditional induction knockout, namely IP3R-tKO for short, and naming a control mouse as IP3R-Con; And 3, finally, injecting adult IP3R-tKO and IP3R-Con into abdominal cavity for 5 days to induce the Cre enzyme to play a recombination effect, obtaining a specific mouse with the three subtypes of IP3R knocked out from mature sperms through genotype identification and recombination efficiency measurement, and finally obtaining a sperm dysfunction type low fertility target model mouse through fertility test.
  2. 2. A gene-edited mouse model obtained by the construction method according to claim 1.
  3. 3. Use of the gene-edited mouse model according to claim 2 in drug screening for improving fertility.
  4. 4. The use according to claim 3, for the study of the mechanisms of diseases associated with dystocopheromone due to disturbance of the IP3R calcium signal.
  5. 5. Use of the gene-edited mouse model according to claim 2 for calcium-reservoir function in mature sperm.
  6. 6. The use according to claim 5, wherein the study of IP3R calcium signaling in sperm is constructed by screening for agonist drugs and testing on a test platform.
  7. 7. The use according to claim 5, wherein Bradykinin and Thimerosal are capable of significantly increasing the level of calcium signaling response of the mouse sperm head calcium pool.
  8. 8. The use according to claim 6, wherein Bradykinin and thermo-al are used as evaluation tool drugs for the abnormality of calcium pool function in human sperm.

Description

Gene editing mouse model construction method for researching calcium library function and low fertility in mature sperm and application thereof Technical Field The invention relates to the technical field of animal model construction, in particular to a gene editing mouse model construction method for researching calcium library function and low fertility in mature sperms and application thereof. Background Reproductive health problems have become a social problem of increasing concern in modern society, and about 6 couples are faced with fertility disorders, wherein about half of male factors are present. The major factors in men are from abnormal spermatogenesis and dyssperm function. The research of abnormal spermatogenesis has a large number of animal models, and the research of dysfunctional spermatozoa is greatly weakened due to the popularization of auxiliary reproduction technologies, because more than half of infertility caused by dysfunctional spermatozoa can be solved by auxiliary reproduction including in vitro fertilization, single sperm injection in egg cytoplasm and the like. However, assisted reproduction is a technique that violates natural conception rules, and there is increasing evidence that offspring obtained by assisted reproduction present various health risks, such as a significantly increased risk of suffering from neurological diseases and cancers. Therefore, the reasons and mechanisms of the mature sperm dysfunction are studied, so that the risk of the birth of the part of patients can be greatly reduced through natural conception or intrauterine injection, and the birth spirit and economic burden of the patients can be reduced. Ca 2+ as an intracellular second messenger is widely involved in various physiological processes such as gene expression control, synaptic transmission, cell differentiation, cell proliferation, cell death, etc. of cells. Research shows that calcium signals are also involved in spermatogenesis, sperm function regulation, fertilization and other events, and are critical to male fertility. Sperm are cells that are "inert" in both gene transcription and translation, and their physiological functions are mainly achieved by intracellular second messengers, enzymatic reactions, post-translational modifications of proteins, etc., where calcium signals are also the regulatory factors of other pathways, and thus are at the heart in the regulation of sperm function. The existing research evidence is based on functional analysis of CatSper calcium channels, and proves that calcium signal increase caused by external calcium influx mediated by the CatSper channels under physiological stimulus is an essential link for fertilization, and the deficiency of the main subunit of the CatSper can lead to sterility due to sperm dysfunction. However, calcium signaling is not only affected by external calcium influx, but also release and recovery of calcium stores, etc. are important processes for regulating calcium signaling, but have not been fully studied since there is a lack of ideal research tool drugs or animal models. In addition, since mature sperm lose a large number of organelles, the function of calcium stores is mainly carried by acrosomes and small numbers of mitochondria, and a large number of proteins regulating calcium stores in somatic cells are lost or lose functions in sperm. However, the IP3R channel is thought to be present in mature sperm and may be involved in the hyperactivated motility of sperm, but there is insufficient evidence, so that, starting from the important and complex intracellular calcium channel of IP3R, the study of the role of calcium stores in sperm function regulation and fertilization is of great importance. IP3R, the 1,4, 5-triphosphate inositol receptor, is a calcium ion channel localized in the endoplasmic reticulum and sarcoplasmic reticulum. Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol, 4-bisphosphate (PIP 2) to form IP3 (inolitol 1,4,5-trisphosphate (IP 3)) and DAG (diacylglycerol). IP3 is delivered intracytoplasmically and the opening of the regulatory channels transmits calcium signals after binding to IP3R receptors located on the sarcoplasmic reticulum or endoplasmic reticulum. There are three different types of IP3R channels, which are highly homologous, and different subtypes can form functional hetero-tetramer channels, so that the IP3R channels have more diversity and are beneficial to participate in wide physiological activity regulation, however, the compensatory effect exists between functions of different channels, and great challenges are brought to analyzing the functions of a single IP3R channel in a specific tissue organ. Furthermore, inhibitors of the IP3R channel 2-APB and Xestospongin C show potential therapeutic effects in neurodegenerative diseases (such as alzheimer's disease) and cardiovascular diseases. Future drug development is striving to find IP3R modulators with high specific