B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J

Stock number: 007914
Product name: Ai14 , Ai14D or Ai14(RCL-tdT)-D
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Ai14 is a Cre reporter tool strain designed to have a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the Gt(ROSA)26Sor locus. Ai14 mice express robust tdTomato fluorescence following Cre-mediated recombination. This strain is congenic on the C57BL/6J genetic background.

The Ai14 allele is very similar in design to the Ai9 allele (Stock No. 007909) - differing only in the absence (Ai14) or presence (Ai9) of an att site-flanked neo selection cassette at the 3' end of the targeted allele.

Importantly, both Ai14 and Ai9 may exhibit low levels of tdTomato expression prior to exposure to Cre recombinase - but the tdTomato expression levels after Cre recombination are greater than those baseline levels. As such, it is recommended that researchers include Cre-negative controls to establish the baseline tdTomato levels in their experiments. See Detailed Description for additional details.

Detailed description

Ai14 mice homozygous for this Rosa-CAG-LSL-tdTomato-WPRE conditional allele (also called Ai14D, Ai14(RCL-tdT)-D or Rosa-CAG-LSL-tdTomato-WPRE::ΔNeo) are viable and fertile. A loxP-flanked STOP cassette is designed to prevent transcription of the red fluorescent protein variant tdTomato (see below). When bred to mice that express Cre recombinase, the resulting offspring will have the STOP cassette deleted in the cre-expressing tissue(s) - resulting in robust tdTomato fluorescence. Because this CAG promoter-driven reporter construct is inserted into the Gt(ROSA)26Sor locus, robust tdTomato expression is determined by which tissue(s) express Cre recombinase. These Ai14 mice are useful as a Cre reporter strain - expressing tdTomato fluorescence following Cre-mediated recombination.

Importantly, the donating investigator reports that very low levels of tdTomato expression may be present prior to introduction of Cre recombinase - but the tdTomato expression levels after Cre recombination are significantly greater than those baseline levels. As such, it is recommended that researchers include Cre-negative Ai14 controls to establish the baseline tdTomato levels in their experiments.

For characterization information, see images at the Allen Institute for Brain Science website (Ai14 images).

The Allen Institute for Brain Science website has specific characterization information for several Cre Driver and Cre Reporter lines. Please see their website for images of Allen Institute for Brain Science experiments performed with all lines.

The Ai14 allele is very similar in design to the Ai9 allele (Stock No. 007909) - differing only in the absence (Ai14) or presence (Ai9) of an att site-flanked selection cassette at the 3' end of the targeted allele. Specifically, the Ai14 allele (Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}) is designed as Rosa26::CAG::FRT::loxP-STOP-loxP::tdTomato::WPRE::polyA::attL, whereas the Ai9 allele (Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}) is designed as Rosa26::CAG::FRT::loxP-STOP-loxP::tdTomato::WPRE::polyA::attB-PGK-FRT-neo-polyA-attP.

Development

The Rosa-CAG-LSL-tdTomato-WPRE targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), tdTomato sequence (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 polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. This entire construct 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 (clone Ai9) were then transiently transfected with a PhiC31-expressing plasmid. The resulting ES cells (clone Ai14) with the PGK-FRT-Neo-polyA cassette deleted (leaving a single attL site) were injected into recipient blastocysts. High percentage chimeric males were sent to The Jackson Laboratory. Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) to establish the mutant colony (as Stock No. 007908). Next, Ai14 mice were further backcrossed with C57BL/6J inbred mice using a marker-assisted approach to generate this congenic colony (Stock No. 007914).

Because this Ai14 conditional allele has the attB/attP-flanked sequences deleted, it may also be called Ai14D, Ai14(RCL-tdT)-D or Rosa-CAG-LSL-tdTomato-WPRE::deltaNeo.

In 2017, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on cohorts from the live colony at The Jackson Laboratory Repository. All of the markers were C57BL/6J allele-type with the following exceptions. The most distal SNP marker on Chromosome 6 [~108 Mbp] consistently shows as 129 allele-type for Ai14 homozygous mice, which is likely a result of its proximity to the Ai14 mutant locus [Gt(ROSA)26Sor at ~113 Mbp]. Some mice had also shown SNP markers on Chromosome 18 segregating for 129 allele-type, but such mice were not used for further breeding units. Since then, SNP testing performed multiple times each year have shown the Chromosome 18 SNP marker as C57BL/6J allele-type on all tested cohorts.

Allele

Symbol: Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}
Name: targeted mutation 14, Hongkui Zeng
MGI accession ID: MGI:3809524
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: Ai14; Ai4; Gt(ROSA)26Sor_tm14(CAG/LSL_tdTomato)Hze; Gt(ROSA)26Sor^{tm14(CAT-tdTomato)Hze}; Gt(ROSA)26Sor^{tm9.1(CAG-tdTomato)Hze}; R26^Tom; R26R^T; R26-RFP; RCL-tdT; Rosa26^Tdt; Rosa26-tdTomato; Rosa-CAG-LSL-tdTomato-WPRE::deltaNeo; Rosa-LSL.dTm; Rosa-lsl-tdTomato; STOP-RFP; tdTomato; Tomato^flox
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: RFP,Red Fluorescent Protein,coral
Site of expression: A loxP-flanked STOP cassette is designed to prevent transcription of the red fluorescent protein variant tdTomato. Upon exposure to Cre recombinase, robust tdTomato fluorescence may be observed. Importantly, the donating investigator reports that very low levels of tdTomato expression may be present prior to introduction of Cre recombinase - but the tdTomato expression levels after Cre recombination are significantly greater than those baseline levels.
Molecular note: The Rosa-CAG-LSL-tdTomato-WPRE targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), tdTomato sequence (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 polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. phiC31-recombination of Gt(ROSA)26Sortm9(CAG-tdTomato)Hze removed the neomycin resistance cassette.