CRISPR-Cas9 system-mediated knockout method for mouse FGF5 (Fibroblast Growth Factor 5) gene

A mouse and construction method technology, applied in the fields of molecular biology and animal genetics and breeding, can solve problems such as unreported research and achieve the effect of improving safety

Inactive Publication Date: 2019-05-28
INNER MONGOLIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] There is no report on the knockout

Method used

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  • CRISPR-Cas9 system-mediated knockout method for mouse FGF5 (Fibroblast Growth Factor 5) gene
  • CRISPR-Cas9 system-mediated knockout method for mouse FGF5 (Fibroblast Growth Factor 5) gene
  • CRISPR-Cas9 system-mediated knockout method for mouse FGF5 (Fibroblast Growth Factor 5) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Optimization of CRISPR-Cas9 vector

[0029] The CRISPR-Cas9 expression vector purchased from Beijing Zhongyuan Company was optimized. The nucleotide sequence of the optimized CRISPR-Cas9 vector (i.e. hCas9 plasmid) is shown in SEQ ID NO: 1, and the hCas9 plasmid map is shown in figure 1 .

Embodiment 2

[0030] Example 2 Construction of sgRNA expression vector

[0031] According to the mouse FGF5 gene sequence (Gene ID 14176), the sgRNA sequence was designed for the exon 1 sequence of FGF5, and the sgRNA expression vector based on the CRISPR-Cas9 system was constructed. The sgRNA expression vector includes four parts: U6 promoter, target sequence, sgRNA backbone and termination signal. Among them, the DNA sequence of the sgRNA action site is as follows: 5'-GAAGGGCAACCCGCGCCTCC-3' (sgRNA target site)

[0032] Use biological software to design the sgRNA sequence according to the sgRNA action site (sgRNA1 target site), clone it into the PMD-19T vector, transform Escherichia coli, pick a single colony after plating, perform bacterial liquid PCR, and identify it by electrophoresis and sequencing. A single colony with correct sequencing was inoculated in LB medium containing Amp, sterilized overnight at 37°C and 220 rpm, the extracted plasmid was named 19T-sgRNA-FGF5, and it was us...

Embodiment 3

[0033] Example 3 Screening of target sites and detection of excision efficiency

[0034] According to the screening results of mouse fetal fibroblast transfection conditions, the optimal transfection condition was determined to be plasmid 10ug; electric shock conditions were voltage (V) 160, pulse time (s) 5 for FGF5-sgRNA, cas9 transfection. According to the molecular weight calculation of FGF5-sgRNA and cas9, ensure that the molar numbers of the final transfected sgRNA and cas9 are basically the same, the sgRNA is slightly more than cas9, and the total DNA amount is guaranteed to be 10ug, and the DNA mixed solution added is not more than 10ul. We diluted the concentration of sgRNA to 200ng / ul; the concentration of cas9 was diluted to 2000ng / ul as the electroporation reaction solution. Finally, 5.5ul of sgRNA; 4.5ul of cas9 were used for transfection.

[0035] Use Premier 5 software to design a pair of PCR primers across different target sites (as shown in Table 1), and carry ...

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Abstract

The invention discloses a CRISPR-Cas9 system-mediated knockout method for a mouse FGF5 (Fibroblast Growth Factor 5) gene. The method comprises the following steps: constructing an sgRNA expression vector based on a CRISPR-Cas9 system according to a mouse FGF5 gene sequence; injecting an sgRNA-Cas9 plasmid mixture into a germ cell pronucleus; performing embryo transplantation and strain gene identification to obtain a genetically mutated mouse; performing PCR (Polymerase Chain Reaction) sequencing and confirmation to obtain a hereditary mouse mutant model finally. A built CRISPR-Cas9-mediated targeting vector provides a simple, convenient, rapid and safe way for the knockout of the mouse FGF5 gene. The method realizes site-directed integration of exogenous gene cell lines without any screening markers, greatly improves the safety of transgenic animals, and has important value in genetic breeding of mice.

Description

technical field [0001] The invention relates to the fields of molecular biology and animal genetic breeding, in particular to a method for knocking out the mouse FGF5 gene mediated by a CRISPR-Cas9 system. Background technique [0002] The CRISPR / Cas9 system is an adaptive immunity that is self-generated by bacteria and archaea through long-term evolution to effectively resist the invasion of foreign DNA. The modification of the type II CRISPR / Cas system by humans has made it another efficient site-specific modification of the genome since Zinc finger nucleases (ZFNs) and TALEN nucleases (Transcription activator-like effector nucleases, TALENs). In this system, mRNA (CRISPR RNA) and CRISPR-associated proteins (CRISPR-associated proteins, Cas proteins) transcribed from clustered regularly interspaced short palindromic repeats (CRISPR) are artificially modified into sgRNA (small guide RNA) and Cas9 protein. The CRISPR / Cas9 system recognizes the target sequence through sgRNA ...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/89C12N15/877A01K67/027
Inventor 郭旭东白宇张晓枫张蒙梁浩
Owner INNER MONGOLIA UNIVERSITY
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