Construction method and application of mir3061 gene rosa26 knock-in heterozygous mouse model

A technology of mouse model and construction method, applied in other methods of inserting foreign genetic material, stably introducing foreign DNA into chromosomes, using vectors to introduce foreign genetic material, etc., can solve problems such as lack and restriction of effective drug development of POF

Active Publication Date: 2021-12-24
SHANGHAI GERIATRIC INST OF CHINESE MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of transgenic mouse models targeting ovarian granulosa cells, the research on the mechanism of POF and the development of effective drugs are severely restricted.

Method used

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  • Construction method and application of mir3061 gene rosa26 knock-in heterozygous mouse model
  • Construction method and application of mir3061 gene rosa26 knock-in heterozygous mouse model
  • Construction method and application of mir3061 gene rosa26 knock-in heterozygous mouse model

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Experimental program
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Effect test

Embodiment 1

[0046] The present invention utilizes CRISPR / Cas9 technology to obtain a method for constructing a Rosa26 site-specific knock-in heterozygous mouse model capable of conditionally overexpressing the Mir3061 gene.

[0047] (1) Experimental animals

[0048] 5-week-old healthy C57BL / 6 mice (weight 18±2g), 100 male and 100 each, provided by Shanghai Southern Model Biotechnology Co., Ltd., can reach the 6-week-old required by the experiment after one week of isolation; 60 healthy ICR mice aged 6 to 8 weeks, half male and half female, weighing 24±2g, were purchased from Shanghai Southern Model Biotechnology Co., Ltd. All mice were fed in an SPF-grade environment and managed in strict accordance with the animal breeding environment According to regulations, the daily temperature is guaranteed to be 22°C, the humidity is 40% to 60%, and the light time and dark time in the animal room are divided into half and half. All relevant operations and handling of animal experiments strictly fol...

Embodiment 2

[0125] 1. Experimental animals

[0126] 5-week-old healthy C57BL / 6 mice (weight 18±2g), 100 male and 100 each, provided by Shanghai Southern Model Biotechnology Co., Ltd., can reach the 6-week-old required by the experiment after one week of isolation; 60 healthy ICR mice aged 6 to 8 weeks, half male and half female, weighing 24±2g, were purchased from Shanghai Southern Model Biotechnology Co., Ltd. All mice were fed in an SPF-grade environment and managed in strict accordance with the animal breeding environment According to regulations, the daily temperature is guaranteed to be 22°C, the humidity is 40% to 60%, and the light time and dark time in the animal room are divided into half and half. All relevant operations and handling of animal experiments strictly follow the relevant management regulations of experimental animals.

[0127] 2. Experimental reagents

[0128] Cas9 Nuclease (NEB USA); Premix TaqTM PCR Enzyme (TAKARA Japan); DNA Purification and Recovery Kit (Tianji...

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Abstract

The invention discloses a construction method and application of a Rosa26 knock-in heterozygous mouse model of the Mir3061 gene. The mouse gene of the construction is Rosa26 LSL / + , the construction method provided by the present invention for the first time has not been reported in any domestic and foreign literature. Provide an effective experimental animal model for the study of the pathogenesis of POF, drug development and efficacy evaluation. The invention also provides the application of the mouse model in screening and preparing drugs for detecting / treating reproductive development diseases, and drugs for detecting / treating aging and apoptosis diseases of ovarian granulosa cells. The mouse model constructed by the method of the present invention provides an effective experimental animal model for genetics of reproductive development and preparation of drugs for detection / treatment of ovarian diseases and related drug screening, has good practical value, and has good medical clinical application prospects and large social benefits.

Description

technical field [0001] The present invention relates to biotechnology, in particular to a construction method and application of a Rosa26 site-specific knock-in heterozygous mouse model capable of conditionally overexpressing Mir3061 gene by using CRISPR / Cas9 technology. Background technique [0002] Premature ovarian failure (POF) is one of the main causes of female infertility, which is largely related to the aging and apoptosis of ovarian granulosa cells, which directly leads to the decline in the quality of follicles at all levels. Due to the lack of transgenic mouse models targeting ovarian granulosa cells, the research on the mechanism of POF and the development of effective drugs are severely restricted. The present invention finds that miR-3061 significantly reduces expression in POF mouse ovarian granulosa cells through RNA-seq high-throughput sequencing. In vitro experiments also confirmed that overexpression of miR-3061 can significantly promote the aging and apo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/85A01K67/027
Inventor 刘特郑锦陈川郁志华林佳佳
Owner SHANGHAI GERIATRIC INST OF CHINESE MEDICINE
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