Gt(ROSA)26Sor^{tm1(CAG-Ncre/vvd*,-vvd*/Ccre,-mKate2)Yzwa}

The targeting vector was designed with (from 5' to 3') a ubiquitously expressed CAG (CMV enhancer/chicken beta-actin) promoter, a frt-flanked STOP cassette (containing a neomycin resistance [neo] cassette and a polyadenylation [pA] signal). A loxP-flanked cassette including PA-Cre 3.0 followed by a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and a pA site were placed downstream of the frt-flanked STOP cassette. PA-Cre 3.0 consists of a split Cre (59/60) with each fragment complemented with nMag or pMag (respectively) and a nuclear localization sequence (NLS) (CreN59-nMag-NLS and NLS-pMag-CreC60), separated by P2A self-cleaving peptide sequence (2A). The nMag and pMag are part of Magnets protein derived from the fragments of photoreceptor gene product Vivid (VVD) from a filamentous fungi Neurospora crassa. This floxed cassette is followed by a nuclear-localized red fluorescent protein (mKate2) followed by another pA signal. This targeting vector was inserted into the Gt(ROSA)26Sor locus. The construct was electroporated into 129Sv/Ev x C57BL/6 derived embryonic stem (ES) cells (clone A20) and correctly targeted ES cells were injected into blastocysts. (J:292311)