Abstract
When transgenic mice are created by microinjection of DNA into the pronucleus, the sites of DNA integration into the mouse genome cannot be predicted. Most methods based on polymerase chain reaction (PCR) that have been used for determining the integration site of foreign DNA into a genome require specific reagents and/or complicated manipulations making routine use tedious. In this report we demonstrate the use of a PCR-based method—TAIL-PCR (Thermal Asymmetric Interlaced PCR) which relies on a series of PCR amplifications with gene specific and degenerate primers to reliably amplify the integration sites. By way of example, using this approach, three separate integration sites were found (on chromosomes 8, 15 and 17) in one transgenic founder. As the sites on chromosomes 8 and 15 failed to segregate in any subsequent progeny, whole chromosome paints were done to determine if translocations involving chromosomes 8 and 15 occurred at the time of transgene integration. Whole chromosome painting could not detect translocations, suggesting that the rearrangements likely involve only small stretches of chromosomes. Site-specific primers were used to identify the progeny carrying only one integration site; these mice were then used as sub-founders for subsequent breedings. Integration site specific primers were used to distinguish homozygous progeny from heterozygotes. TAIL-PCR thus provides an easy and reliable way to (1) identify multiple integration sites in transgenic founders, (2) select breeders with one integration site, and (3) determine zygosity in subsequent progeny. Use of this strategy may also be considered to map integration sites in situations of unexpected phenotype or embryonic lethality while creating new transgenic mice.
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Acknowledgements
This work was supported by the National Institutes of Health (K08 DK073707 to M.M.P. and R01 HL062923 to B.T.S.). The core facilities used for the work were supported in part by P30 CA015704 (FHCRC Comprehensive Cancer Center Grant). The authors would like to acknowledge Nanyan Jiang of the transgenic core for production of the transgenic mice, employees of the animal health resources (AHR) for maintenance of colonies, and the genomics core for automated DNA sequencing, Bonnie Larson and Helen Crawford for assistance with formatting the manuscript (all at FHCRC), Dr. Elaine Raines, University of Washington, Seattle, WA (for the LZRS-SIN-CD68L-HA-EGFP vector), Dr. Hermann Bujard, University of Heidelburg, Germany for the pUHRT62-1 vector) and Dr. Daphne Preuss, University of Chicago, Chicago, IL (for suggestions regarding TAIL-PCR optimization).
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Pillai, M.M., Venkataraman, G.M., Kosak, S. et al. Integration site analysis in transgenic mice by thermal asymmetric interlaced (TAIL)-PCR: segregating multiple-integrant founder lines and determining zygosity. Transgenic Res 17, 749–754 (2008). https://doi.org/10.1007/s11248-007-9161-4
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DOI: https://doi.org/10.1007/s11248-007-9161-4