B6;129S-Rorb^{tm1.1(cre)Hze}/J

Stock number: 023526
Product name: Rorb-IRES2-Cre-D
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

Rorb-IRES2-Cre-D knockin mice have Cre recombinase expression directed to Rorb-expressing cells, without disrupting endogenous RAR-related orphan receptor beta expression. These mice may be useful for studying this nuclear hormone receptor/DNA-binding protein in hormone-responsive gene expression and behavior.

Detailed description

The Rorb-IRES2-Cre-D (also called Rorb-IRES2-Cre-Δ or Rorb-IRES2-Cre-Δneo/hygro) targeted mutation has an IRES2 sequence and a Cre recombinase gene inserted downstream of the translational STOP codon of the RAR-related orphan receptor beta gene (Rorb). As such, Rorb-IRES2-Cre-D mice have both endogenous gene and Cre recombinase expression directed to Rorb-expressing cells by the endogenous promoter/enhancer elements of the RAR-related orphan receptor beta locus. When Rorb-IRES2-Cre-D mice are bred with mice containing loxP-flanked sequences, Cre-mediated recombination will result in deletion of the floxed sequences in Rorb-expressing cells in the offspring.

The donating investigator reports Cre recombinase expression (in situ hybridization using a Cre-specific probe) is observed in a pattern very similar to that of the endogenous Rorb gene. Specifically, cre expression is strong in the zonal layer of the superior colliculus and subregions of thalamus. Dense, patchy cre expression is seen in cortical layer 4, with sparse cre expression in cortical layer 5 and 6. Cre recombinase is also expressed in trigeminal nucleus and small patches of cells in cerebellum. The donating investigator did not examine cre expression in tissues other than brain.

Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator reports homozygous mice are viable but demonstrate an abnormal walking gait with an exaggerated high step. Breeding homozygous mice together produces some pups with more severe phenotype (runty and tremulous).

For characterization information, see images at the Allen Institute for Brain Science website (Rorb-IRES2-Cre images).

Development

These Rorb-IRES2-Cre-D mice (also called Rorb-IRES2-Cre-Δ or Rorb-IRES2-Cre-Δneo/hygro) have an IRES2 sequence and a Cre recombinase gene inserted immediately downstream of the translational STOP codon of the RAR-related orphan receptor beta gene (Rorb). The specific details are below.

The targeting vector contained, from 5' to 3', a partial Rorb sequence from intron 9 (with an frt3), a partial Rorb exon 10 sequence up to and including the endogenous stop codon, an internal ribosome entry site 2 (IRES2) sequence (allows translation initiation in the middle of an mRNA sequence), a Cre recombinase gene, a bovine growth hormone polyA sequence, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, an frt5 site, an mRNA splice acceptor, the 3' portion of the hygromycin gene with SV40 late 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-IRES2-Cre-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 transgene was removed). In 2013, males from generation N4F1 were sent to The Jackson Laboratory Repository. Upon arrival, males were used to cryopreserve sperm. To establish the living Rorb-IRES2-Cre-D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Allele

Symbol: Rorb^{tm1.1(cre)Hze}
Name: targeted mutation 1.1, Hongkui Zeng
MGI accession ID: MGI:5507855
Generation method: Targeted
Attributes: Recombinase-expressing
Marker symbol: Rorb
Marker name: RAR-related orphan receptor beta
Chromosome: 19
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: cre expression is strong in the zonal layer of the superior colliculus and subregions of thalamus. Dense, patchy cre expression is seen in cortical layer 4, with sparse cre expression in cortical layer 5 and 6. Cre recombinase is also expressed in trigeminal nucleus and small patches of cells in cerebellum.
Molecular note: The targeting vector contained, from 5' to 3', a partial Rorb sequence from intron 9 (with an frt3), a partial Rorb exon 10 sequence including the endogenous stop codon, an internal ribosome entry site 2 (IRES2) sequence, a Cre recombinase gene, a bovine growth hormone polyA sequence, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, an frt5 site, an mRNA splice acceptor, the 3' portion of the hygromycin gene with SV40 late polyA signal, and an AttP site. This construct was electroporated into G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Chimeric mice were bred to PhiC31-expressing mice 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-IRES2-Cre-D mice were bred with C57BL/6J wildtype mice to remove PhiC31 transgene.