Analysis of transgene integration sites in transgenic pigs by fluorescence in situ hybridization.
The production of pigs transgenic for human decay accelerating factor (hDAF) as potential donors for clinical organ xenotransplantation was reported several years ago. For this purpose it is required that high levels of hDAF are expressed at relevant sites in transplantable organs. Currently, homozygous lines have been produced as well as lines from crosses between heterozygous animals from different founder lines, termed 'jigsaw' pigs. The purpose of the 'jigsaw' crosses is to combine the desirable hDAF protein expression patterns found in different founder lines. Initial selection of the 'jigsaw' pigs is based on the inheritance of the hDAF integration sites from both lines. Litters with potential homozygous transgenics and 'jigsaw' transgenics were analysed by fluorescence in situ hybridization (FISH) and slot blot analysis. Results show that both slot blot analysis and FISH are suitable to distinguish between pigs that are heterozygous and homozygous fir hDAF. However, FISH has the advantage of producing results more rapidly. For the identification of 'jigsaw' pigs FISH analysis was required since slot blot analysis lacked the required accuracy. On basis of these results, FISH analysis was made part of the routine screening programme for hDAF transgenic pigs.
Kuipers
H W
HW
Department of Surgery, University of Cambridge, UK.
Langford
G A
GA
White
D J
DJ
D000818:Animals; D030801:Animals, Genetically Modified; D001360:Avidin / Q000737:chemistry; D018958:CD55 Antigens / Q000235:genetics / Q000378:metabolism; D003433:Crosses, Genetic; D015342:DNA Probes; D004727:Endothelium / Q000378:metabolism; D005260:Female; D019085:Fluorescent Antibody Technique, Direct / Q000379:methods; D019084:Fluorescent Antibody Technique, Indirect; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008297:Male; D008677:Metaphase; D013552:Swine / Q000235:genetics*; D014018:Tissue Distribution; D019076:Transgenes
D018958:CD55 Antigens; D015342:DNA Probes; D001360:Avidin