title: A novel genotyping strategy based on allele-specific inverse PCR for rapid and reliable identification of conditional FADD knockout mice.
abstract: The apoptotic adapter protein FADD has been shown to play diverse roles in cell survival and proliferation. FADD knockout embryos died of heart defects, rendering Cre/loxP-mediated conditional FADD knockout mice a unique tool for investigating FADD-dependent nonapoptotic mechanism. Previously, these genetically engineered mice were identified by time-consuming Southern blot or controversial real-time PCR. In this article, we report a novel genotyping strategy based on allele-specific inverse PCR (ASI-PCR) for rapid and reliable identification of conditional FADD knockout mice. In this strategy, the knockout nature of FADD was simply identified by screening the absence of the wild type FADD-specific ASI-PCR product. Using this method, we accurately identified CD4-Cre-mediated T cell specific FADD knockout mice. The whole process can be accomplished in any normal biological laboratory within 12 h using genomic DNA from tail biopsy. The proposed ASI-PCR-based approach is simple, rapid, sensitive, reproducible, and especially suitable for genotyping small amount of spatiotemporally restricted biopsies and large animal population. We believe that the strategy described in this article may be of general utility in genotyping other conditional gene knockout mice.
authors.affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, College of Life Sciences, Nanjing University, Nanjing, 210093, PR China.
authors.forename: Xiangbai
authors.initials: X
authors.lastname: Dong
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.forename: Shufeng
authors.initials: S
authors.lastname: Li
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Zi-chun
authors.initials: ZC
authors.lastname: Hua
mesh_terms: D000483:Alleles; D000818:Animals; D015139:Blotting, Southern; D053200:Fas-Associated Death Domain Protein / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D005838:Genotype; D051379:Mice; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000378:metabolism; D013997:Time Factors
chemical_list: C505534:Fadd protein, mouse; D053200:Fas-Associated Death Domain Protein