A targeting vector was designed to insert an internal ribosome entry site (IRES), a Cre recombinase sequence, an SV40 polyA signal, and an frt-flanked neo cassette into the 3' untranslated region (after the translational termination site) of the Gad2 (glutamic acid decarboxylase 2) gene. This construct was electroporated into C57BL6/S129 hybrid embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient tetraploid blastocysts. Chimeric mice were bred with Actin-FLPe mice (on a C57BL/6 congenic background (N10)) to remove the selection cassette. The FLPe transgene was subsequently bred out of the background.