Analysis of transgenic animal pedigrees using the junction-polymerase chain reaction.

Division of Molecular Immunology, and Neurobioiogy, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada MSG 1XS Here we describe a modificat ion of the polymerase chain reaction--junctionPCR IJ-PCR)--that has been designed to facilitate the identification and subsequent screening of transgenic animals and their offspring. This procedure targets a unique recombinat ion junction, which is present in the concatamerized transgene insert, for amplification. Although several nonradioactive PCR-based strategies have recently been described for analyzing transgenic mice, 11,21 each suffers from the possibility of identifying falsepositive mice due to contaminat ing plasmid DNA. Since J-PCR involves ampl i fying a recombinat ion junction generated by the concatamerizat ion of transgene inserts, the interpretation of the amplif icat ion reaction is not affected by overt levels of contaminat ing plasmid DNA. As shown in Figure 1A, a defined junction is generated upon transgene concatamerizat ion and insertion. In animals that carry more than one copy of the transgene, this junction represents a unique DNA fragment that can be the target of J-PCR analysis using appropriately posit ioned primers. The location of these primers allows amplification of both circular plasmid DNA (pMBP/p72MAG) and of linear transgenic DNA such that two discrete and diagnostic bands are produced. J-PCR analysis produces a 3.2-kb band from plasmid DNA or a 0.9-kb band from transgene-positive DNA (see Fig. lc). We have used this procedure successfully to verify the mult icopy nature of several transgenic lines established using the vector outl ined in Figure 1 (unpublished). Specifically, the founder mice represented in lanes b, c, d, and f of Figure 1C were positive by the J-PCR analysis (note the 0.9-kb band), whereas mouse e tested negative by both Southern analysis of tail biopsy DNA and by J-PCR (Fig. 1C,D). Amplification of circular plasmid DNA failed to generate the diagnostic 0.9-kb fragment (lanes h and i), demonstrat ing