Integration site analysis in transgenic mice by thermal asymmetric interlaced (TAIL)-PCR: segregating multiple-integrant founder lines and determining zygosity.
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.
Pillai
Manoj M
MM
Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N., Seattle, WA 98109-1024, USA.
Venkataraman
Gopalakrishnan M
GM
Kosak
Steven
S
Torok-Storb
Beverly
B
D000818:Animals; D015703:Antigens, CD / Q000502:physiology; D015214:Antigens, Differentiation, Myelomonocytic / Q000502:physiology; D015139:Blotting, Southern; D001947:Breeding; D033481:Chromosomes, Mammalian / Q000235:genetics*; D005260:Female; D018703:Founder Effect; D006580:Genetic Carrier Screening; D006720:Homozygote; D008297:Male; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011995:Recombination, Genetic; D019076:Transgenes / Q000502:physiology*; D014178:Translocation, Genetic / Q000235:genetics; D000097382:CD68 Molecule
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C081026:CD68 protein, mouse; D000097382:CD68 Molecule