These PRimary colours In the MEmbrane (PRIME) mice have a CAG promoter followed by three pairs of heterospecific lox sites (loxP, lox2272 and loxN), and the PRIME construct, all targeted into the Gt(ROSA)26Sor locus (RosaPRIME). Specifically, the RosaPRIME construct contains three pairs of heterospecific lox sites that separate 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), and 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 is 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. In the presence of Cre recombinase, mice express either M-mSc-I or M-mTq2 or M-mVen. The membrane localization emphasizes the cell and tissue geometry making these mice useful for 3D lineage tracing and tracking, yielding additional color combinations when two different fluorescent proteins are expressed from the two alleles. The donating investigator reports that in practice each of the three colors should combine in a 1:1:1 ratio, yet the mVenus recombination events occur less often. They also report that only the iRFP670 has be detected in the targeted ES cells by fluorescence microscopy or FACS so far, but not yet in tissues. The M-mSc-I, M-mTq2, and M-mVen fluorophores are efficiently detected by FACS and fluorescent microscopy in both the targeted ES cells and primary tissues. Mice homozygous for the RosaPRIME allele are viable and fertile with no abnormal phenotype. No leakiness of M-mSc-I, M-mTq2, or M-mVen expression has been detected prior to cre recombination.

 Complete characterization of PRIME mice is available as a PhD thesis with permission from the donating investigator, Dr. Ren&eacute;e van Amerongen [<a href=" https://pure.uva.nl/ws/files/140294841/Chapter_3.pdf">PRIME: A novel multicolour reporter for lineage tracing, cellular visualisation and cell segmentation (2023)</a>].

These PRimary colours In the MEmbrane (PRIME) mice have a CAG promoter followed by three pairs of heterospecific lox sites (loxP, lox2272 and loxN), and the PRIME construct, all targeted into the Gt(ROSA)26Sor locus (RosaPRIME). PRIME contains four fluorescent proteins and functions as a Cre recombinase-inducible reporter of either a nuclear near-infrared fluorescent protein (3xNLS-iRFP670) or one of three membrane-bound fluorescent proteins from a single genomic locus.

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