Construction method of Mir3061 gene Rosa26 fixed-point knock-in heterozygote mouse model and application thereof

A technology of mouse model and construction method, applied in other methods of inserting foreign genetic materials, stably introducing foreign DNA into chromosomes, using vectors to introduce foreign genetic materials, etc., can solve the problems of restricting the development and lack of effective drugs for POF , to achieve the effects of good medical clinical application prospects, great social benefits, and great application value

Active Publication Date: 2019-06-28
SHANGHAI GERIATRIC INST OF CHINESE MEDICINE
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  • Description
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  • Application Information

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Problems solved by technology

Due to the lack of transgenic mouse models targeting ovarian granulosa cells, the rese...

Method used

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  • Construction method of Mir3061 gene Rosa26 fixed-point knock-in heterozygote mouse model and application thereof
  • Construction method of Mir3061 gene Rosa26 fixed-point knock-in heterozygote mouse model and application thereof
  • Construction method of Mir3061 gene Rosa26 fixed-point knock-in heterozygote mouse model and application thereof

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Experimental program
Comparison scheme
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Embodiment 1

[0050] 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.

[0051] (1) Experimental animals

[0052] 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 of a Rosa26 gene Mir3061 fixed-point knock-in heterozygote mouse model and an application thereof. The constructed mouse gene is Rosa26LSL/+, and the construction method is provided for the first time and is not reported in any domestic or foreign literature reports. And the model provides an effective experimental animal model for the research on thepathogenesis of POF, the research and development of medicines and the evaluation of drug effect. The invention also provides the application of the mouse model in screening and preparing medicines for detecting/treating reproductive development diseases and medicines for detecting/treating aging and apoptosis diseases of ovarian granulosa cells. The model provides an effective experimental animalmodel for reproductive development genetics, preparation of medicines for detecting/treating ovarian diseases and screening of related medicines, and has good practical value, good medical clinical application prospect and great social benefit.

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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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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