These Ai66(RCRL-tdT)Δneo mice (also called Ai66(RCRL-tdT)-D, Ai66Δneo, or Ai66D) have both a Rox-flanked STOP cassette and a loxP-flanked STOP cassette upstream of the tdTomato fluorescent protein, all inserted into the Gt(ROSA)26Sor locus. The specific details are below.
The Rosa-pCAG-RSR-LSL-tdTomato-WPRE-bGHpA-AttB-PGK-Neo-polyA-AttP targeting vector was designed with (from 5' to 3')
a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS),
an Rox-flanked STOP cassette (with a short open reading frame, followed by translational stops in all three reading frames, a synthetic polyA sequence, a polyA sequence from the human growth hormone gene, and a polyA sequence from the Herpes Simplex Virus TK gene),
a loxP-flanked STOP cassette (with stop codons in all three reading frames linked to three repeats of the SV40 polyA signal),
a sequence encoding the tdTomato fluorescent protein (a non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein (tandem dimer)),
a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability),
a bovine growth hormone polyA signal,
an AttB site,
a PGK promoter-Neomycin resistance gene-PGK polyA cassette,
and an AttP site.
This entire targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were selected.
Chimeric mice were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to remove the AttB/AttP-flanked PGK-Neo-polyA cassette and replace it with the recombined AttB/AttP site (AttL). The resulting Ai66(RCRL-tdT)Δneo mice were bred with C57BL/6J wildtype mice for at least two additional generations (and the PhiC31 gene was removed) prior to sending to The Jackson Laboratory Repository in 2013. Upon arrival, males were used to cryopreserve sperm. To establish the living Ai66(RCRL-tdT)Δneo mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
