CRISPR/Cas9 technology-mediated site-specific integration method of goat VEGF gene

A fixed-point integration and goat technology, which is applied in the fields of molecular biology and animal genetics and breeding, to achieve the effects of delaying the regression period, improving gene editing efficiency, and increasing cashmere production

Inactive Publication Date: 2018-08-21
INNER MONGOLIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The method of using CRISPR/Cas9 system to mediate the site-sp...

Method used

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  • CRISPR/Cas9 technology-mediated site-specific integration method of goat VEGF gene
  • CRISPR/Cas9 technology-mediated site-specific integration method of goat VEGF gene
  • CRISPR/Cas9 technology-mediated site-specific integration method of goat VEGF gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Optimization of Cas9-gRNA expression vector

[0034] The Cas9-gRNA expression vector purchased from Beijing Weishang Lide Biotechnology Co., Ltd. was optimized. The nucleotide sequence of the optimized Cas9-gRNA expression vector is shown in SEQ ID NO: 1, and the plasmid map is shown in figure 1 .

Embodiment 2

[0035] Example 2 Construction of Cas9-gRNA expression vector

[0036] According to the goat FGF5 gene sequence (NC_030813.1), the gene sequence is shown in SEQ ID NO:2. A gRNA sequence was designed on the exon 1 sequence of the FGF5 gene, and a gRNA expression vector based on the CRISPER / Cas9 system was constructed. The gRNA expression vector includes four parts: U6 promoter, target sequence, gRNA backbone and termination signal. Goat VEGF gene-directed knock-in model diagram based on CRISPR / Cas9 system see figure 2. Use biological software to design the sgRNA sequence according to the sgRNA action site, and the sequence is shown in SEQ ID NO:3. Synthesize Target-Sense and Target-Anti, whose sequences are shown in SEQ ID NO: 6, 7, anneal to form a partially complementary oligo dimer, and then insert the oligo dimer into the Cas9 / gRNA expression vector to obtain a complete The Cas9 / gRNA expression vector was transformed into Escherichia coli Trans1-T1, plated, and after 9 ...

Embodiment 3

[0037] Example 3 Transfection of Goat Fetal Fibroblasts by Electroporation

[0038] The primary goat fetal fibroblasts were thawed and cultured in a 100mm culture dish, and the medium was changed every other day. Before transfection by electroporation, the cells should be passaged once to improve their growth status, so as to improve the survival rate and transfection efficiency of cells after transfection. After subculture, when the cell growth confluence reaches 80% to 90%, first wash away residual metabolites and residual serum in the culture medium with PBS buffer, add 2 mL of 0.25% trypsin to digest for 2 min, and wash with 10% FBS containing The DMEM / F12 culture medium was digested, and the cells were collected in a 15 mL centrifuge tube by gently blowing and mixing with a pipette gun, centrifuged at 800 rpm for 5 min, washed twice by adding Opti-MEM and discarding the supernatant. In order to ensure the transfection efficiency, an appropriate amount of Opti-MEM can be ...

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Abstract

The invention discloses a method for completing site-specific integration of a goat VEGF gene through mediation of a CRISPER/Cas9 system. In the method, according to a goat FGF5 gene sequence, a CRISPER/Cas9 system-based Cas9-gRNA expression vector and a CRISPER/Cas9 system-based VEGF targeting vector are constructed; then, the optimized Cas9-gRNA expression vector and the optimized VEGF targetingvector are co-transfected into a goat fetal fibroblast to obtain a cell strain in which the VEGF gene is integrated at a specific FGF5 site. The constructed CRISPER/Cas9-based targeting vector provides a simple, fast and safe way for the site-specific integration of the goat VEGF gene and targeted knockout of an FGF5 gene. By the method, any screening marker gene is not involved in the cell strain screening process, so that the safety of a transgenic animal is greatly improved. Therefore, the method has an important value in genetic goat breeding and research on gene functions.

Description

technical field [0001] The invention relates to the fields of molecular biology and animal genetic breeding, in particular to a method for site-directed integration of goat VEGF gene and knockout of FGF5 gene mediated by CRISPR / Cas9 technology. Background technique [0002] Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR), as the third-generation gene editing technology, is known for its unprecedented specificity, effectiveness and versatility. It is derived from the acquired immune system of prokaryotes, provides acquired immunity to resist the invasion of foreign gene elements such as phages, and widely exists in archaea and prokaryotes. The CRISPR / Cas9 system has recently developed into an effective tool for specific editing of eukaryotic genomes. Compared with traditional zinc finger nucleases (znicfinger nucleases, ZFNs) and transcription activator-like effector nucleases (transcriptionactivator-like effector nucleases, TALENs), the CRISPR / Cas9 system...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N15/877C12N5/10A01K67/027
CPCA01K67/0275A01K2217/072A01K2227/102A01K2267/02C07K14/485C12N5/0603C12N5/0656C12N9/22C12N15/8509C12N15/8772C12N15/907C12N2510/00C12N2800/107C12N2810/10
Inventor 呼啸刘东军郝斐高源梁浩
Owner INNER MONGOLIA UNIVERSITY
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