High‐throughput fluorescent screening of transgenic animals: Phenotyping and haplotyping

Abstract

Methods for genotyping transgenic animals currently consist of extracting genomic DNA from blood or tissue followed by PCR or Southern blot analysis. These methods when used to screen large numbers of animals can be time consuming and expensive. Therefore, we developed a novel method that allows high-throughput screening of phenotypic changes on leukocytes, resulting from the transgenic genotype. This technique allows investigators to quickly screen a large number of animals without the need to extract DNA from each one. Moreover, since blood is collected for the initial screening, putative homozygotes can be confirmed by conventional methods using the same blood samples. We collected blood from wild-type alphagal positive and alphagal knockout mice and probed for the presence of Galalpha(1-->3)Gal (alphagal) epitopes. Also, alloantigen specific antibodies were used to determine the haplotype of our outbred mouse colony in order to develop an inbred line. alphagal epitopes were detected in wild-type but not alphagal knock-out samples. To validate these results, PCR was used to demonstrate the native alphagal gene in wild-type and the pGKneo construct in alphagal knock-out mice. Furthermore, haplotypes were determined and mice divided for backcrosses. This screening method is useful for both preliminary screening of transgenic mice and the development of an inbred mouse colony by rapid determination of MHC I haplotype. Here, we demonstrate the use of this technique and show how it can be a valuable tool, saving time and resources in both investigator effort and animal husbandry.