Method for knocking out CRISPR/Cas9 mediated sheep FGF5 gene and integrating MTNR1A gene at fixed point

A gene and gene editing technology, applied in the field of genetic engineering, can solve problems such as slow progress in sheep fecundity and low fecundity of high-quality meat varieties, and achieve the effect of avoiding abnormal gene function and biological safety

Inactive Publication Date: 2019-06-11
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The fecundity of high-quality meat breeds is low, and the progress of improving sheep fecundity through traditional purebred breeding and improved hybridization is slow

Method used

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  • Method for knocking out CRISPR/Cas9 mediated sheep FGF5 gene and integrating MTNR1A gene at fixed point
  • Method for knocking out CRISPR/Cas9 mediated sheep FGF5 gene and integrating MTNR1A gene at fixed point
  • Method for knocking out CRISPR/Cas9 mediated sheep FGF5 gene and integrating MTNR1A gene at fixed point

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of CRISPR / Cas9 Targeting Vector

[0046] The targeting site is located in the third exon of the sheep FGF5 gene (NM_001246263.2), and the corresponding targeting site is as follows figure 1 As shown, wherein, the nucleotide sequence identifying the target in the sgRNA sequence is shown in SEQ ID NO: 1, and the DNA sequence encoding the above-mentioned sequence is shown in SEQ ID NO: 2. According to the above target sequence, the corresponding primer sequence was designed and synthesized by Sangon Biotechnology Co., Ltd., and the purification method was HPLC. The specific sequence is shown in Table 1.

[0047] Table 1 Goat FGF5 gene targeting sequence primers

[0048] Nucleotide name

sequence (5'-3')

Ovis aries-FGF5-sgRNA-F

caccgAGGTTCCCCTTTCCGCACCT

Ovis aries-FGF5-sgRNA-R

aaacAGGTGCGGAAAGGGGAACCTc

[0049] Formation of Olio nucleic acid sequence: Dilute primers to 100 μM, phosphorylation anneal, system: Ov...

Embodiment 2

[0051] The construction of embodiment 2 donor vector

[0052] Such as image 3 As shown, in order to insert MTNR1A into the FGF5 target site, a gene locus with a homology arm needs to be inserted into the target site by means of Homology directed repair (HDR). Since the construction of the donor vector requires cloning four long fragments into a destination vector, coupled with the uncertainty of the restriction site, it is difficult to ensure that each fragment can be spliced ​​properly, so Gibson Assembly based on homologous recombination was used , which can stitch together more long fragments efficiently and seamlessly without restriction of enzyme cutting sites.

[0053] The construction process of the donor vector is as follows: Figure 4 As shown, the upstream homology arm 5-HA, CYP17-promoter, MTNR1A(MT1)-bGH poly(A) signal, and the downstream homology arm 3-HA need to be ligated into the backbone vector pUC57-Amp, where the upper, The downstream homology arm and CY...

Embodiment 3

[0061] Example 3 Cell Transfection

[0062] 1. Prepare fetal fibroblasts by conventional methods.

[0063] 2. Adopt nuclear transfer method.

[0064] with nucleofection-Amaxa TM Basic Nucleofector TM Kit for Primary Mammalian Fibroblasts (Lonza), using Nucleofector TM The program V-024 of 2b was used for transfection, 10 micrograms of the donor vector and 10 micrograms of the targeting vector. For the transfection steps, please refer to the kit instructions. For transfection see Image 6 .

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Abstract

The invention provides a method for knocking out a CRISPR / Cas9 mediated sheep FGF5 gene and integrating an MTNR1A gene at a fixed point.The method includes the steps of transferring the CRISPR / Cas9 targeting vector of the specific targeting third exon of the sheep FGF5 gene and the gene homologous recombination vector into a sheep fetus fibroblast to obtain a sheep FGF5 gene knockout cell with a fixed-point integration of the MTNR1A gene. The donor vector and the targeting vector are co-transfected into sheep fetus fibroblasts in a nuclear transfer mode, and specific expression of MTNR1A in sheep ovary tissue can be achieved through a CYP17 promoter in the unmarked donor vector constructed through a Gibson Assembly method. The separated and identified positive monoclonal cell strain can beused as a nuclear donor for nuclear transplantation of somatic cells to obtain cloned embryos, so that a solid foundation is laid for culturing transgenic clone sheep which locally strengthens melatonin signals and produces more hairs in a reproductive system.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for CRISPR / Cas9-mediated knockout of the sheep FGF5 gene and site-specific integration of the MTNR1A gene. Background technique [0002] Reproductive traits are important economic traits in the breeding industry, and effectively improving the reproductive traits of sheep is one of the keys to improving the level of sheep breeding. The fecundity of high-quality meat breeds is low, and the progress of improving sheep fecundity through traditional purebred breeding and improved hybridization is slow. In recent years, scholars at home and abroad have done a lot of research on the molecular genetic basis of sheep, looking for and locating the main genes that affect the reproductive mechanism of sheep at the molecular level, laying the foundation for accurate breeding improvement. Candidate genes for high fecundity found so far include: BMPR-1B, BMP15, GDF9, and m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N15/12C12N5/10A01K67/027
Inventor 刘国世李广栋张鲁连正兴吕东颖姚昱君吴昊朱天奇姬鹏云
Owner CHINA AGRI UNIV
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