B6.Cg-Rorb^{tm2.1(tTA2)Hze}/J

Stock number: 028537
Product name: Rorb-T2A-tTA2-D
Research areas: Neurobiology Research, Research Tools

Description

Rorb-T2A-tTA2-D knockin mice are designed to have both endogenous gene and synthetic modified tetracycline-regulated transactivator (tTA2^S) gene expression directed to Rorb-expressing cells by the endogenous promoter/enhancer elements of the RAR-related orphan receptor beta locus. These Rorb-T2A-tTA2-D mice are designed to be a Tet-Off tool that allows dox-conditional expression of genes in layer 4 of cortex.

Detailed description

The RAR-related orphan receptor beta protein encoded by the Rorb gene is a DNA-binding protein from the NR1 subfamily of nuclear hormone receptors.


The Rorb-T2A-tTA2-D knockin mutation has an in-frame insertion of a viral 2A oligopeptide (T2A) and a synthetic modified tetracycline-regulated transactivator (tTA2^S), all in place of the Rorb translational STOP codon. This is designed to have both endogenous gene and tTA2 expression directed to Rorb-expressing cells by the endogenous promoter/enhancer elements.

When Rorb-T2A-tTA2-D mice are bred with mice carrying a gene of interest under the regulatory control of a tetracycline-responsive promoter element (TRE, TetRE or tetO), expression of the target gene in the Rorb-expressing cells may be conditionally abolished with administration of the tetracycline analog doxycycline (dox) in the double mutant offspring. Specifically, the donating investigator reports that Rorb-T2A-tTA2-D activates tetO-regulated gene expression is restricted to layer 4 of cortex. The donating investigator did not examine tTA2^S activity in tissues other than brain.

Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator did not examine tTA2^S activity in tissues other than brain. To date (December 2015), it has not been attempted to make this strain homozygous.

For characterization information, see images at the Allen Institute for Brain Science website (Rorb-T2A-tTA2 images).

Development

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 (tTA2^S) 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 (tTA2^S) 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).

Allele

Symbol: Rorb^{tm2.1(tTA2)Hze}
Name: targeted mutation 2.1, Hongkui Zeng
MGI accession ID: MGI:5784556
Generation method: Targeted
Attributes: Transactivator
Marker symbol: Rorb
Marker name: RAR-related orphan receptor beta
Chromosome: 19
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Site of expression: These mice are designed to be a Tet-Off tool that allows dox-conditional expression of genes in layer 4 of cortex.
Molecular note: The targeting vector contains, from 5' to 3',a partial Rorb intron 9 containing an FRT3 site, a partial Ndnf exon 10 sequence up to (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 (tTA2) gene, 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. PhiC31-mediated recombination removed the AttB/AttP-flanked sequence (PGK-Neo-polyA::FRT5::RNA splice acceptor::3'hygro-polyA) and replaced it with the recombined AttB/AttP site (AttL).