〔Abstract〕 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 construc- ted 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 se- quencing , 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 fu- ture research .