title: Increased Specific Labeling of INS-1 Pancreatic Beta-Cell by Using RIP-Driven Cre Mutants with Reduced Activity.
abstract: Ectopically expressed Cre recombinase in extrapancreatic tissues in RIP-Cre mice has been well documented. The objective of this study was to find a simple solution that allows for improved beta-cell specific targeting. To this end, the RIP-Cre and reporter CMV-loxP-DsRed-loxP-EGFP expression cassettes were configurated into a one-plasmid and two-plasmid systems, which labeled approximately 80% insulin-positive INS-1 cells after 48 h transfection. However, off-target labeling was robustly found in more than 15% insulin-negative Ad293 cells. When an IRES element was inserted in front of Cre to reduce the translation efficiency, the ratio of recombination between INS-1 and Ad293 cells increased 3-4-fold. Further, a series of Cre mutants were generated by site-directed mutagenesis. When one of the mutants, Cre(H289P) in both configurations, was used in the experiment, the percentage of recombination dropped to background levels in a number of insulin-negative cell lines, but decreased only slightly in INS-1 cells. Consistently, DNA substrate digestion assay showed that the enzymatic activity of Cre(H289P) was reduced by 30-fold as compared to that of wild-type. In this study, we reported the generation of constructs containing RIP and Cre mutants, which enabled enhanced beta-cell specific labeling in vitro. These tools could be invaluable for beta-cell targeting and to the study of islet development.
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong, 515063, China.
authors.forename: Gen-cheng
authors.initials: GC
authors.lastname: Gong
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong, 515063, China.
authors.forename: Wen-zhu
authors.initials: WZ
authors.lastname: Fan
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong, 515063, China.
authors.forename: Di-zheng
authors.initials: DZ
authors.lastname: Li
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong, 515063, China.
authors.forename: Xiong
authors.initials: X
authors.lastname: Tian
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong, 515063, China.
authors.forename: Shao-jun
authors.initials: SJ
authors.lastname: Chen
authors.affiliation: Laboratory of Cell Senescence, Shantou University Medical College, Shantou, Guangdong, 515041, China.
authors.forename: Yu-cai
authors.initials: YC
authors.lastname: Fu
authors.affiliation: Department of Endocrinology, the First Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, 515041, China.
authors.forename: Wen-can
authors.initials: WC
authors.lastname: Xu
authors.affiliation: Multidisciplinary Research Center, Shantou University, Shantou, Guangdong, 515063, China.
authors.forename: Chi-ju
authors.initials: CJ
authors.lastname: Wei
mesh_terms: D000818:Animals; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000235:genetics*; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D051381:Rats; D013194:Staining and Labeling; D014162:Transfection
chemical_list: D007328:Insulin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases