B6;129S-Gt(ROSA)26Sor^{tm66.1(CAG-tdTomato)Hze}/J

Stock number: 021876
Product name: Ai66(RCRL-tdT)-D or Ai66D
Research areas: Neurobiology Research, Research Tools

Description

Ai66(RCRL-tdT)Δneo (also called Ai66(RCRL-tdT)-D, Ai66Δneo, or Ai66D) is a dual Dre/Cre recombinase fluorescent reporter. This Rosa-CAG-RoxSTOPRox-LoxSTOPLox-tdTomato::deltaNeo conditional allele is designed with both a Rox-flanked STOP cassette and a loxP-flanked STOP cassette preventing transcription of the tdTomato fluorescent protein. After removal of the flanked STOP cassettes by Dre- and Cre-recombination, tdTomato fluorescence is expected in cells/tissues expressing both Dre recombinase and Cre recombinase.

Detailed description

Ai66(RCRL-tdT)Δneo mice (also called Ai66(RCRL-tdT)-D, Ai66Δneo, or Ai66D) harbor the Rosa-CAG-RoxSTOPRox-LoxSTOPLox-tdTomato::deltaNeo conditional allele designed with both a Rox-flanked STOP cassette and a loxP-flanked STOP cassette preventing transcription of the tdTomato fluorescent protein. Although under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread expression of tdTomato is prevented by both STOP cassettes. After removal of both flanked STOP cassettes via dre- and cre-mediated recombination, expression of tdTomato is expected in cells/tissues where the expression patterns of the individual promoters driving Dre recombinase and Cre recombinase overlap.

Specifically, the donating investigator reports that Ai66(RCRL-tdT)Δneo mice have tdTomato fluorescence in cells expressing both Dre and Cre recombinase (via breeding to Dre and Cre recombinase-expressing mice or via recombinant adeno-associated virus injections [AAV-synapsin promoter-Cre and AAV-synapsin promoter-Dre expression plasmids]). tdTomato expression is presumably also detectable by mRNA [in situ hybridization] and antibody staining/immunohistochemistry. No significant tdTomato expression is observed prior to introduction of both Dre recombinase or Cre recombinase. Extremely rare fluorescence was reported in some Cre+/Dre-/Ai66(RCRL-tdT)Δneo mice (1-3 cells within the entire cortex). The donating investigator also reports that order in which the flanked STOP cassettes are removal is arbitrary. Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator has not attempted to generate homozygous mice to date (April 2013).

For characterization information, see images at the Allen Institute for Brain Science website (Ai66(RCRL-tdT) images).

Development

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).

Allele

Symbol: Gt(ROSA)26Sor^{tm66.1(CAG-tdTomato)Hze}
Name: targeted mutation 66.1, Hongkui Zeng
MGI accession ID: MGI:3809505
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Site of expression: Expression of tdTomato fluorescence depends upon promoter used that drives both FLP and Cre recombinase.
Molecular note: 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. PhiC31-mediated recombination removed the AttB/AttP-flanked PGK-Neo-polyA cassette and replace it with the recombined AttB/AttP site (AttL).