The Rorb-T2A-tTA2-D targeted mutation (also called Rorb-2A-tTA2-Δ or Rorb-2A-tTA2-Δneo/hygro) has a viral 2A oligopeptide (T2A) that mediates ribosomal skipping and a synthetic modified tetracycline-regulated transactivator (tTA2S) gene inserted in place of the translational STOP codon of the RAR-related orphan receptor beta gene (Rorb) on chromosome 19. The specific details are below.
The targeting vector contained, from 5' to 3',
a partial Rorb intron 9 containing an frt3 site,
a partial Ndnf exon 10 sequence up (but not including) the endogenous stop codon,
a T2A sequence that is in-frame with the Rorb coding sequence,
a synthetic modified tetracycline-regulated transactivator (tTA2S) gene,
a bovine growth hormone polyA signal,
an AttB site,
a PGK-Neo-polyA cassette,
an frt5 site,
an mRNA splice acceptor,
the 3' portion of the hygromycin gene with SV40 polyA signal,
and an AttP site.
This construct was electroporated into (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Chimeric mice were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to remove the AttB/AttP-flanked sequences (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replace it with the recombined AttB/AttP site (AttL).
The resulting Rorb-T2A-tTA2-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 transgene was removed). In 2016, black male mice from generation N6 were sent to The Jackson Laboratory Repository. Upon arrival, males are used to cryopreserve sperm. To establish the living Rorb-T2A-tTA2-D mouse colony, an aliquot of the frozen sperm is used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).
