Rapid screening of transgenic type II and type XI collagen knock-out mice with three-primer PCR.

We demonstrate in this article the rapid determination of heterozygous and homozygous transgenic knock-out mice using the polymerase chain reaction (PCR) with three primers. A similar technique was described by Horton et al. (3), which demonstrated threeprimer PCR in the genotype determination of knock-out RAG-2 immunodeficient mice. However novel this approach may be, one must conclude that the procedure by Horton is quite time-consuming. This is a rate-limiting step when utilizing transgenic/knockout mice as a biological tool; most knock-out mice develop fatal phenotypes at birth or within a few hours post-partum. We offer in this article an expeditious approach for screening knock-out mice, for determining the genotype and for achieving satisfactory results within the same day of pup mice birth through the use of three-primer PCR. The knock-out mice in this study are each deficient for a specific collagen gene. In the collagen II knock-out mouse, there is a deletion of the COL2A1 gene, which has been inactivated or “knocked out” by insertion of a neomycin-resistance gene. The heterozygous genotype of these mice develops normally, but the homozygous offspring develops a fatal phenotype within minutes after birth (4). In the collagen XI knock-out mouse, there is also a replacement for a portion of the gene with the neomycin-resistance gene in the collagen gene Col11A2. The heterozygous phenotype of these mice are normal, but the homozygotes develop limited phenotypes such as bulging eyes and a short snout accompanied by a short body stature. The phenotypes are within themselves an aid in the determination of the genotype, but this does not hold true as in the case of the heterozygous variety of both type II and type XI knock-out mice. To avoid any ambiguity, PCR is the ultimate tool utilized in the determination of the mice genotype. Furthermore, we have found that the use of three primers (Figure 1) in PCR analysis uniquely distinguishes between a heterozygous or homozygous genotype of the mouse of interest. Additionally, this proposed technique serves as an internal control when certain PCRs are prone to yielding nonspecific amplified DNA.