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Construction and application of inducible uterine epithelium specific genetic engineering mouse

A genetic engineering and specific technology, which is applied in the construction and application field of inducible uterine epithelial-specific genetic engineering mice, can solve uterine development problems, gene deletion, gene function research and other problems, and achieve the effect of accelerating the breeding speed

Active Publication Date: 2021-10-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006]Wnt7a-Cre: It is expressed in the fetal period. If the target gene plays an important role in the fetal uterus, it may cause uterine development problems. It cannot express the gene in the adult uterus function in normal physiological processes
[0007] The Cre sequences of the above genetically engineered mice all replace the position of the corresponding gene, resulting in the deletion of the gene, so only heterozygotes (Cre / +) can be used

Method used

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  • Construction and application of inducible uterine epithelium specific genetic engineering mouse
  • Construction and application of inducible uterine epithelium specific genetic engineering mouse
  • Construction and application of inducible uterine epithelium specific genetic engineering mouse

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1) Genomic DNA extraction steps are as follows:

[0049] 1. Use ear tag pliers and matching ear tags to mark the ears of the mice, cut a small piece of tail tissue about 0.2cm long and put it into a 1.5ml EP tube, and write the corresponding ear tag on the EP tube. label.

[0050] 2. Add 200 μl of rat tail lysate and 4 μl of proteinase K (invitrogen) at a concentration of 20 mg / ml to each EP tube, mix well, and centrifuge so that the tail tissue is submerged in the mixture, and place in a metal heater at 60°C for 3 hours, After cooling at 95°C for 15 minutes, centrifuge to take the supernatant, which is the mouse genomic DNA solution.

[0051] 2) Agarose gel electrophoresis:

[0052] Preparation of 2% agarose gel: Add 120mL 1×TEA working solution and 2.4g agar powder into a conical flask, heat in a microwave oven until the agarose is completely dissolved, add 6 μL nucleic acid dye (Sangon Biotech) according to the ratio of 1:2000 Engineering), shake well and pour it ...

Embodiment 2

[0064] Wnt7a rtTA / + TeO + / + wxya F / + The construction method is mainly divided into two steps:

[0065] S1: first genotype mouse Wnt7a rtTA / + and TetO cre / + Hybridization, the genotype of the first generation is Wnt7a rtTA / + TeO cre / + The probability is 1 / 4, then the genotypes are all Wnt7a rtTA / + TeO cre / + A pair of male and female mice crossed to obtain the genotype Wnt7a rtTA / rtTA TeO cre / + The probability of is 1 / 8.

[0066] S2: Separate the genotypes to mTmG F / F and Wnt7a rtTA / rtTA TeO cre / + A pair of male and female mice crossed, the genotype in the offspring is Wnt7a rtTA / + TeO cre / + wxya F / + The mice can be used for dox-inducible experiments, and the genotype is Wnt7a rtTA / + TeO + / + wxya F / + mice were used as control mice.

Embodiment 3

[0068] On the 7th day after birth, the newborn rats were injected into the uterine cavity, and DOX 2 μl was given in the uterine cavity of the newborn rats at a concentration of 10 mg / ml. Among them, the intrauterine injection operation of offspring is the key point of the technology, because the uterine cavity of offspring is too small, and the offspring are weak, and they are still in the lactation stage. The surgical method and postoperative recovery process of offspring need to be carefully and repeatedly tested to determine the best plan. . After repeated exploration and practice, the following experience was obtained:

[0069] First, in the selection of anesthetics, we chose ether, which is less toxic and easy to operate. Surgical experience shows that the pups of PND7 can start the operation after being anesthetized with ether for about 1 minute, and the operation process is controlled at about 7 minutes.

[0070] Second, separate the mother rat and the female offsprin...

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Abstract

The invention discloses construction and application of an inducible uterine epithelium specific genetic engineering mouse. A Wnt7a rtTA / + inducible uterine epithelium specific gene engineering mouse is constructed by knocking an rtTA gene expression sequence behind a Wnt7a gene sequence of the mouse at a fixed point, the Wnt7a rtTA / + inducible uterine epithelium specific gene engineering mouse is hybridized with a TetO cre / + mouse and a target gene Loxp mouse, a Tet-On system can be activated after DOX is given, Cre enzyme is expressed, and the target gene is knocked out. The gene of interest can be knocked out or overexpressed in mouse uterine epithelial cells in a space-time controllable manner, so that the function of the gene in uterine epithelium is researched. The technology can also be used for tracking the cell lineage in the mouse uterine epithelium growth and differentiation process through hybridization with a reporter gene mouse. In addition, a homozygous mouse (rtTA / rtTA) is also used, so that the breeding speed of the target mouse is increased.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction and application of an inducible uterine epithelial-specific genetic engineering mouse. Background technique [0002] The construction of uterine epithelial-specific genetically engineered mice mainly includes the following types: [0003] In 2014, the Ltf-Cre genetically engineered mouse edited by the Sudhansu K. Dey laboratory is a genetically engineered mouse that specifically expresses Cre enzyme in the uterine cavity epithelium and uterine glands, but its Cre enzyme is only fully developed when the mouse is an adult. Express. [0004] Sprr2f-Cre (Small proline-rich protein 2f) is also a uterine epithelial knockout gene mouse, but the specificity is not good, and leaky expression occurs in the cerebellum and kidney; Wnt7a-Cre is also a uterine epithelial knockout gene mouse, but the same , gene editing also occurred in the hair follicle epithelium. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027A61D7/00
CPCA01K67/0278A01K67/0275A61D7/00A01K2217/072A01K2217/15A01K2227/105A01K2267/03
Inventor 苏仁伟吴瑶刘晓征
Owner SOUTH CHINA AGRI UNIV
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