CRISPR/Cas9 technology mediated down producing goat VEGF gene fixed-point knock-in method

A cashmere goat, gene technology, applied in the field of animal molecular breeding, can solve the problem of lack of integrity

Active Publication Date: 2018-09-25
INNER MONGOLIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of CRISPR / Cas9 technology in cashmere goat molecular breeding still lacks a complete technical solution, and many technical problems need to be solved, such as improving the efficiency of gene editing, selecting appropriate editing sites and designing precise and efficient sgRNA, and integrating exogenous Gene template construction to avoid off-target effects, screening, identification, and amplification of exogenous gene-specific integration cells, using positive cells to construct reconstructed embryos through Somatic cell Nuclear Transplantation (SCNT) technology, and reconstructing embryos to develop in vitro , embryo transfer, embryo transfer and pregnancy management of pregnant ewes, postpartum lamb management, identification and management of offspring cashmere goats with fixed-point integration of exogenous genes, etc.

Method used

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  • CRISPR/Cas9 technology mediated down producing goat VEGF gene fixed-point knock-in method
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  • CRISPR/Cas9 technology mediated down producing goat VEGF gene fixed-point knock-in method

Examples

Experimental program
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Effect test

Embodiment 1

[0115] Example 1: Design of gRNA based on cashmere goat CCR5 full-length genome sequence (Gene ID: 102178672) Design of gRNA based on target sequence is a basic requirement of CRISPR / Cas9 technology. For site-directed integration of foreign genes, the selection of target sequences is very important, which involves the normal growth and development of cells, embryos and individuals and the expression of foreign genes, so the selection of target sites is creative.

[0116] The present invention selects the cashmere goat CCR5 gene as the target site, and not only refers to the literature data, but also conducts sequence characteristic analysis on the cashmere goat CCR5 full-length genome sequence (Gene ID: 102178672). The first exon of the cashmere goat CCR5 gene is short, only 112bp, the second exon is longer, 1312bp, and the start codon is on the second exon, so the present invention is based on the second exon of CCR5. Sequences Four gRNAs (gRNA1, gRNA2, gRNA3 and gRNA4) with ...

Embodiment 2

[0117] Example 2: Construction of gRNA expression vector

[0118] The construction of gRNA expression vector requires corresponding originals, including promoter, gRNA sequence and backbone sequence, transcription termination signal, etc., which constitute a transcription cassette of gRNA sequence and backbone sequence. Therefore, the construction of gRNA expression vector requires creativity.

[0119] The schematic diagram of the structure of the gRNA plasmid designed in the present invention is shown in Figure 2, including the U6 promoter, gRNA and its backbone sequence and termination signal sequence. First follow figure 1 As shown, the 5'-end (318bp) fragment of gRNA and the 3'-end (117bp) fragment of gRNA were obtained by overlapping PCR, and finally a 455bp fragment encoding the gRNA and its backbone sequence was obtained (Figure 8). Then, using the gRNA vector as the basic backbone, an expression vector pCCR5-gRNAs that includes U6 promoter, gRNA and its backbone seq...

Embodiment 3

[0123] Example 3: Detection of gRNA validity

[0124] The pCCR5-gRNA1, pCCR5-gRNA2, pCCR5-gRNA3 and pCCR5-gRNA4 were co-transfected into CFFCS with the hCas9 vector by electroporation, respectively. After 48 h, the cellular genomic DNA was extracted. The DNA extraction steps were completed according to the instructions of the Wizard Genomic DNA Purification Kit.

[0125] Use specific primers (Table 2) designed according to the sequences on both sides of the CCR5-1, CCR5-2, CCR5-3 and CCR5-4 sites for PCR amplification. The PCR reaction system is LATaqTM II 25 μL, upstream primers (10uM / L ) 2μL, downstream primer (10uM / L) 2μL, template 2μL, d3H2O 19μL. PCR reaction conditions: 95°C 5min; 95°C 1min, 57°C 30sec, 72°C 45sec, 33 cycles; 72°C 10min, 16°C 30min. Fragments containing sgRNA target sites were amplified from the cashmere goat genome, detected by electrophoresis, and the target fragments were recovered and purified by gel using the GeneJET Gel Extraction Kit.

[0126] T...

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Abstract

The invention provides a method for finishing down producing goat VEGF gene fixed-point knock-in mediated by a CRISPR/Cas9 system. The method comprises the following steps: establishing a gRNA expression vector and a VEGF homologous recombination vector on the basis of the CRISPR/Cas9 system according to a CCR5 gene sequence of down producing goats; then jointly transferring three optimized vectors into fibroblasts of down producing goat fetuses to obtain VEGF gene fixed-point knocked-in positive cells; and preparing VEGF gene fixed-point integrated down producing goats by using a somatic nuclear transfer technology. The established targeting vector based on the CRISPR/Cas9 system provides a simple, rapid and safe path for fixed-point knock-in of goat VEGF genes. The method does not involve any screening marker genes in a cell line screening process, therefore, the safety of transgenic animals is greatly improved, and the method has high value on research of genetic breeding and gene functions of down producing goats.

Description

technical field [0001] The invention belongs to the field of animal molecular breeding, in particular to a method for obtaining a transgenic cashmere goat by using CRISPR / Cas9 technology to integrate VEGF gene at a specific point. Background technique [0002] In recent biological research, genome editing (Genome editing) technology is the latest research technology for researchers to understand the function of specific genes. With the completion of whole genome sequencing of more and more species, the new challenge facing researchers is how to obtain gene-related function and application information from a large amount of data. Gene editing technology is a powerful research tool to accomplish this important goal, for which it was named one of the top ten important scientific advances by Science in 2012. In recent years, genome-directed editing has developed rapidly. The production of artificial endonuclease (engineered endonuclease, EEN) has changed the limitations in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/85C12N15/877A01K67/027
CPCA01K67/0275A01K67/0276A01K2217/072A01K2217/075A01K2227/102A01K2267/02C07K14/485C07K14/715C12N9/22C12N15/8509C12N15/8772C12N15/907
Inventor 梁红宇刘东军梁浩呼啸王辉王志刚
Owner INNER MONGOLIA UNIVERSITY
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