The RC::RFLTG triple-recombinase responsive indicator allele has a rox-flanked STOP, frt-flanked STOP and loxP-flanked tdTomato::STOP preventing transcription of eGFP. Although under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread expression of either fluorescent protein is prevented by STOP cassettes. After removal of the first two STOP cassettes by Dre- and FLP-recombination, robust tdTomato fluorescence is expected in cells/tissues where the expression patterns of the individual promoters driving dre and Flp overlap. Further exposure to Cre recombinase removes the floxed-tdTomato::STOP, resulting in robust eGFP fluorescence.

Specifically, the donating investigator reports that RC::RFLTG mice have no tdTomato expression prior to introduction of both Dre and FLP recombinases, and no significant eGFP expression prior to introduction of Dre, FLP and Cre recombinases. Although a low level of eGFP expression is consistently present in some cell types prior to cre expression, removal of the floxed-tdTomato::STOP results in significantly greater levels of eGFP expression. The donating investigator reports that the premature eGFP fluorescence is barely visible when imaged using parameters calculated for recombinase-expressing cells. The donating investigator also reports that, for robust tdTomato expression, the order in which the rox-flanked STOP and frt-flanked STOP cassettes are removed is arbitrary.

Because both fluorescent proteins can fill the axons of neurons, the simultaneous tracing of projections from two different neuronal subpopulations is possible.

Mice homozygous for the RC::RFLTG allele are viable and fertile with no reported gross physical or behavioral abnormalities.

dre-, Flp- and/or cre-mediated removal of specific STOP cassettes within the RC::RFLTG allele results in the following derivative alleles:

i. The dual-recombinase responsive indicator allele RC::RLTG (Stock No. 026931) allows dre-inducible tdTomato fluorescence and subsequent cre-inducible eGFP fluorescence.

ii. The dual-recombinase responsive indicator allele RC::FLTG (Stock No. 026932) allows Flp-inducible tdTomato fluorescence and subsequent cre-inducible eGFP fluorescence.

iii. The single-recombinase responsive indicator allele RC::LTG has constitutive tdTomato fluorescence and allows cre-inducible eGFP fluorescence.

iv. The single-recombinase responsive indicator allele RC::RG has no tdTomato expression (tdTomato::STOP sequences removed) and allows dre-inducible eGFP fluorescence.

v. The single-recombinase responsive indicator allele RC::FG has no tdTomato expression (tdTomato::STOP sequences removed) and allows Flp-inducible eGFP fluorescence.