The Camk2a-CreERT2 transgene was designed with the mouse Camk2a promoter, CreERT2 fusion gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), bovine growth hormone polyA signal, and PGK-Puro-SV40polyA cassette. (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells, already targeted with an Ai3 mutation in the Gt(ROSA)26Sor locus, were transfected with the Camk2a-CreERT2 transgene. Correctly targeted ES cells harboring both the Ai3 mutation and Camk2a-CreERT2 transgene were injected into recipient blastocysts. The resulting chimeric animals were bred to C57BL/6J mice to generate the double mutant colony. Subsequent mating with C57BL/6J wildtype mice resulted in offspring harboring only the Camk2a-CreERT2 transgene (wildtype at the Gt(ROSA)26Sor locus).
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