Generation of a FAM50A knockout Beta-TC-6 cell line using CRISPR/Cas9 technology and preparation of a FAM50A polyclonal antibody
10.19405/j.cnki.issn1000-1492.2025.11.016
- VernacularTitle:利用 CRISPR/Cas9 技术构建 FAM50A 基因敲除的Beta-TC-6 细胞系及 FAM50A 的多克隆抗体制备
- Author:
Yaxua Qiu
1
;
Xiangrui Meng
1
;
Xiaoyan Xie
2
;
Sitong Cheng
1
;
Yufan Peng
2
;
Siqi Liu
1
;
Xue Zhao
3
;
Zhangfeng Hu
3
;
Junqiao Xing
3
;
Weihua Wang
3
Author Information
1. School of Medicine ,Jianghan University , Wuhan 430056
2. School of Life Sciences ,Jianghan University , Wuhan 430056
3. School of Life Sciences , Institute of Microalgae Synthetic Biology and Green Manufacturing , Jianghan University , Wuhan 430056
- Publication Type:Journal Article
- Keywords:
FAM50A;
antibody preparation;
gene knockout;
cilia;
pancreas;
diabetes mellitus
- From:
Acta Universitatis Medicinalis Anhui
2025;60(11):2105-2112
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct a Family with sequence similarity 50 member A(FAM50A) gene knockout mouse insulinoma pancreatic β-cell line Beta-TC-6 using CRISPR/Cas9 gene editing technology and to prepare polyclonal antibodies specifically recognizing FAM50A.
Methods:Two guide RNAs(sgRNAs) targeting the FAM50A gene were designed,and a recombinant plasmid expressing blue fluorescent protein(BFP) was constructed for gene knockout.The successfully constructed plasmid was transfected into Beta-TC-6 cells,and BFP-positive single cells were isolated for clonal expansion.The expanded monoclonal cell lines were genotyped by Sanger sequencing,and FAM50A protein expression was assessed by Western blot.Purified human recombinant FAM50A protein was used to immunize New Zealand rabbits for the preparation of a polyclonal antibody.The specificity of the prepared antibody was then validated using the successfully established FAM50A knockout cell line.
Results:A monoclonal cell line with a successful knockout of the FAM50A gene was identified.Sanger sequencing confirmed base deletions at the target site.Western blot analysis showed a complete absence of FAM50A protein expression in this cell line.The prepared polyclonal antibody successfully recognized endogenous murine FAM50A protein in wild-type Beta-TC-6 cells and in hTERT-RPE1 cells overexpressing human FAM50A-GFP fusion protein,while no signal was detected in the FAM50A knockout cells.
Conclusion:This study successfully established a FAM50A gene knockout Beta-TC-6 cell model and generated a FAM50A polyclonal antibody,providing powerful tools for future research.
- Full text:2026030511335405278利用CRISPR_Cas9技术构建FAM50A基因敲...eta-TC-6细胞系及FAM50A的多克隆抗体制备_邱雅萱.pdf
