Figure 1. Gene targeting strategy.

The targeting vector, containing a 13.0 kb fragment of Adora3 isolated from a mouse BAC clone library, was prepared to introduce two LoxP sites into Adora3, one into the 5’ non-coding region proximal to exon 1 and the second into intron 1. This permits Cre recombinase-mediated excision of exon 1 including the ATG start site along with ~2 kb of upstream sequence containing promoter elements. This targeting strategy avoids depletion of the transmembrane and immunoglobulin domain containing 3 splice variant (TMIGD3 or Adora3i3) that shares exon 2 of Adora3 with the A₃AR [42]. Correctly targeted ESCs (line D3 derived from blastocysts of a 129S2/SvPas mouse) were injected into C57Bl/6 blastocysts, generating 60–90% chimeric male mice estimated from coat color that transmitted the floxed allele via germline, providing the genotype Adora3^{LoxP(FrtneoFrt)LoxP/+)}. The neo gene in intron 1 was removed by mating with a mouse expressing the Flp recombinase transgene while leaving the LoxP site behind. A. Restriction map of the LoxP-targeted Adora3 allele. Bars denote Southern blot probes named 5’ probe and 3’ probe. B. Southern blotting of a correctly targeted ESC line used for blastocyst injections. Left panel. Digestion with NheI followed by hybridization with the 5’ probe reveals a 10.8 kb band diagnostic of the floxed allele due to presence of a NheI site within the neo cassette and a 16.6 kb band diagnostic of the wild-type allele. Right panel. Digestion with BglI and hybridization with the 3’ probe yields a 19.5 kb band from the wild-type allele and a 16.6 kb band from the floxed allele created by a BglI site within the neo cassette. An additional Southern blot was performed by hybridizing a BglI digest with a probe corresponding to the neo cassette, which revealed a single 16.6 kb band ensuring that the targeting vector did not randomly insert into non-endogenous targets (not shown). Appropriate targeting was further confirmed by PCR amplification to detect presence of the LoxP sites, the neo cassette, and site-specific insertion (not shown).