Method for constructing mouse ABCC4 gene knocked out by CRISPR/Cas9 technology and application of ABCC4 gene knockout mouse
A gene-knockout mouse technology, which is applied in the field of gene editing, can solve the problems of lack of research and application of chronic obstructive pulmonary disease, and achieve the effects of shortening time, simple operation, and reducing off-target effects
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[0054] A method for constructing ABCC4 systemic knockout mice using CRISPR / Cas9 technology. The specific implementation process mainly includes gRNA target site selection, microinjection and identification of F0 generation mice, homozygous breeding and identification.
[0055] gRNA target site selection
[0056] The Abcc4 gene (NCBI Reference Sequence: NM_001033336.3; Ensembl: ENSMUSG00000032849) is located on mouse chromosome 14 and contains 31 exons in total. The ATG start codon is located in exon 1, and the TGA stop codon is located in exon 31 (Transcript:
[0057] ENSMUST00000036554). Exon 2 starts at 1.89% of the coding region, exons 2 and 3 cover 5.84% of the coding region, and does not contain other known genes, so exon 2 and exon 3 are selected as knockout regions, Such as figure 1 shown. Select gRNA target sites within 2000 bp upstream of exon 2 and within 2000 bp downstream of exon 3 non-tandem repeat sequences, and design and synthesize gRNA, analyze the selecte...
Embodiment approach
[0091] 1. CRISPR / Cas9 technology is the third-generation gene site-specific editing technology, which has become an efficient gene editing tool because of its precise targeted cutting function. However, other gene editing technologies, such as traditional zinc finger nuclease and transcription activator-like effector nuclease gene editing technologies, may also achieve ABCC4 gene knockout.
[0092] 2. The present invention selects exon 2 and exon 3 to design gRNA for knockout. Since the ABCC4 gene contains 31 exons, gRNA may also be designed to knock out the ABCC4 gene at other exons.
[0093] Therefore, due to the diversity and breadth of techniques, there may be different techniques or different implementation methods for the same technique to construct ABCC4 gene knockout mice. However, based on the previous research results, it was found that the SNP site of ABCC4 mutation is related to the risk of COPD, and the present invention discloses for the first time the specific ...
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