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.
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Acknowledgments
This study is supported by the following funds to Zi-chun Hua from the Chinese National Nature Science Foundation (30330530, 30425009, and 30270291) and the Ministry of Education of China (SRFDP 20030284040). The authors are grateful to Dr. Astar Winoto (UC Berkeley, California, USA) for his generous providing of genetically modified mice.
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Dong, X., Li, J., Li, S. et al. A Novel Genotyping Strategy Based on Allele-specific Inverse PCR for Rapid and Reliable Identification of Conditional FADD Knockout Mice. Mol Biotechnol 38, 129–135 (2008). https://doi.org/10.1007/s12033-007-9002-y
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DOI: https://doi.org/10.1007/s12033-007-9002-y