Gt(ROSA)26Sor^{tm1.1(CAG-iRFP*/mScarlet-I*/mTq2*/mVenus*)Rva}

The targeting vector contains (from 5' to 3') a strong CAGG (CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid) promoter, three pairs of heterospecific lox sites (loxp, lox2272 and loxN), and the PRimary colours In the MEmbrane (PRIME) construct (described in greater detail below), followed by an FRT-flanked neomycin resistance (neo) cassette. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation into B6_ColA_RMCE embryonic stem (ES) cells. Flp-mediated recombination removed the FRT-flanked neo cassette. The Rosa(PRIME) construct contains three pairs of heterospecific lox sites that are inserted separating four fluorescent proteins: a nuclear near-infrared fluorescent protein (3xNLS-iRFP670), membrane-bound red fluorescent protein (M-mSc-I), membrane-bound cyan fluorescent protein (M-mTq2), membrane-bound yellow fluorescent protein (M-mVen). iRFP670 is constitutively expressed in all cells. To ensure that only one fluorescent protein is expressed at any one time, an SV40 polyadenylation signal was inserted downstream of each open reading frame. Upon Cre activation, the lox sites recombine to create three mutually exclusive excision possibilities with the fluorescent protein that is placed first after the CAG promoter being expressed. (J:101977)