Homology comparison between human and pig SERPING1 and establishment of pig PFFs with SERPING1 knockout
10.19405/j.cnki.issn1000-1492.2022.04.001
- Author:
Meng Wang
1
;
Xiaohan Zhu
1
;
Xiaorui Liu
1
;
Lin Li
1
;
Ying Wang
1
;
Haiyuan Yang
1
;
Yifan Dai
1
Author Information
1. Dept of Medical Genetics，Nanjing Medical University， Jingsu Key Laboratory of Xenotransplantation，Nanjing 211166
- Publication Type:Journal Article
- Keywords:
SERPING1;
CRISPR / Cas9;
porcine fetal fibroblasts
- From:
Acta Universitatis Medicinalis Anhui
2022;57(4):525-509
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To buildSERPING1-knockout porcine fetal fibroblasts(PFFs) based on CRISPR/Cas9 technology and provide cell experimental materials for the construction of hereditary angioedema models.
Methods:Firstly, protein structure prediction software was used to analyze the amino acid homology between human and pigSERPING1. Secondly, the eighth exon was selected as the knockout target by screening the effective coding region of the pigSERPING1gene. A single guide RNA targeting pigSERPING1was designed, synthesized and linked to pX330 vector containing Cas9 endonuclease. G418 was used to obtain the positive monoclonal cells after transfection into PFFs. Finally, the circumstance of CRISPR/Cas9 mediated knockout was assessed by the T7 EN1 enzyme digestion assay and the genotypes of monoclonal cells were identified by sequencing analysis
Results:Bioinformatic analysis revealed that theSERPING1protein of human and pig had high homology, amino acid sequence identity reached 65.87%. A vector targetingSERPING1was constructed successfully and transfected into cells. Monoclonal cells with knockoutSERPING1gene were obtained through drug screening. Sequencing confirmed the mutant genotype.
Conclusion:The human and pigSERPING1sequences and their protein structures are highly homologous, and it is suitable for the construction of disease model. CRISPR/Cas9 expression vectors were constructed to achieve theSERPING1gene targeting in PFFs.SERPING1-knockout monoclonal cells were obtained, which could contribute to the construction of theSERPING1knockout miniature pig model.
- Full text:2024122615064632422人_猪SERPING1同源...PING1敲除细胞系的建立_王蒙.pdf
