STOCK Notch1^tm2Rko Notch2^tm3Grid Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze} Tg(KRT5-cre/ERT2)2Ipc/JeswJ

Stock number: 030855
Product name: K5CreER;N1^fl;N2^fl;Ai9 [or K5-fl(N1/N2)-Ai9]
Research areas: Cancer Research, Cell Biology Research, Dermatology Research, Developmental Biology Research, Neurobiology Research, Research Tools, Sensorineural Research

Description

K5CreER;N1^fl;N2^fl;Ai9 mice (or K5-fl(N1/N2)-Ai9) are quadruple mutant animals with tamoxifen-inducible Cre recombinase expression directed to basal epithelial cell populations - resulting in Notch1/Notch2 knock-out and robust tdTomato fluorescence. These mice allow inducible genetic manipulations for studying Notch pathway disruption in epithelial tissues (e.g., epidermis, salivary gland, digestive tract, peripheral olfactory system) as well as UV-light skin damage and cancer biomarkers.

Detailed description

The keratin 5 gene (KRT5) encodes cytokeratin-5 or K5, a type II cytokeratin specifically expressed in basal epithelial cells (including epidermis, salivary gland and digestive tract). K5 is a biomarker for several different types of cancer (including breast and lung). Notch signaling pathways are identified in both embryonic and adult development of several organ systems (including nervous, cardiovascular, endocrine and respiratory), as well as in cancer pathways.

K5CreER;N1^fl;N2^fl;Ai9 mice (or K5-fl(N1/N2)-Ai9) are quadruple mutant animals harboring the K5CreER^T2 transgene, Cre-inducible knock-out (floxed) alleles of Notch1 and Notch2 (N1^fl and N2^fl) and the Cre-inducible tdTomato fluorescent reporter allele R26R-LSL-tdTomato (Ai9).

The donating investigator summarizes the phenotype below for mice hemizygous for the transgene and homozygous for all three targeted mutations (K5CreER;N1^fl/fl;N2^fl/fl;Ai9^{lsl-tdT/lsl-tdT}) on a predominantly C57BL/6 genetic background.


Prior to tamoxifen exposure, K5CreER;N1^fl/fl;N2^fl/fl;Ai9^{lsl-tdT/lsl-tdT} mice are viable and fertile with no reported gross physical or behavioral abnormalities. Before induction, normal NOTCH1/NOTCH2 expression is observed (as expected). After tamoxifen exposure, the K5CreER^T2 transgene is designed to eliminate NOTCH1/NOTCH2 expression - however, they report actual recombination rates in the Notch1 or Notch2 loci can vary per animal between 50-100%. As such, they recommend direct confirmation of knock-out be performed. The basal epithelial cell NOTCH1/NOTCH2 knock-out results in atopic dermatitis-like disease (characterized by the degeneration of hair follicles into large cysts).


Some cells exhibit tdTomato fluorescence prior to tamoxifen exposure, but the number of tdTomato fluorescent cells is significantly increased upon tamoxifen introduction. When expressed, tdTomato is observable via direct fluorescence without the need for staining. The donating investigator attributes the pre-tamoxifen tdTomato expression (but normal NOTCH1/NOTCH2 expression) to high levels of K5CreER^T2 expression that allow sporadic entry into the nucleus - resulting in recombination of the more-accessible Gt(ROSA)26Sor locus but not the Notch1 or Notch2 loci. Also note that, as reported for Ai9 single mutant mice (Stock No. Stock No. 007909), 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.


To date (August 2017), the donating investigator has not characterized tamoxifen-inducible Cre recombinase expression in tissues other than skin. In addition, they report mice homozygous for the K5CreER^T2 transgene (as identified by test cross/progeny ratios) are viable and fertile.

These mice allow inducible genetic manipulations for studying Notch pathway disruption in epithelial tissues (e.g., epidermis, salivary gland, digestive tract, peripheral olfactory system) as well as UV-light skin damage and cancer biomarkers.
For example, K5CreER;N1^fl/fl;N2^fl/fl;Ai9^{lsl-tdT/lsl-tdT} mice were used to study the effect of Notch1/Notch2 knock-out in horizontal basal cells (HBCs) of the peripheral olfactory system following an olfactotoxin-induced (MeBr gas) tissue damage/regeneration protocol [Herrick et al. 2017 J Neurosci]. This concluded that canonical Notch signaling determines neuronal versus non-neuronal progenitor cell fate in the regenerating adult olfactory epithelium.

The Cre-ERT2 fusion protein consists of Cre recombinase fused to a triple mutant form of the human estrogen receptor which does not bind its natural ligand (17β-estradiol) at physiological concentrations but will bind the synthetic estrogen receptor ligands 4-hydroxytamoxifen (OHT or tamoxifen) and, with lesser sensitivity, ICI 182780. Restricted to the cytoplasm, Cre-ERT2 can only gain access to the nuclear compartment after exposure to tamoxifen. To counteract the mixed estrogen agonist effects of tamoxifen injections, which can result in late fetal abortions in pregnant mice, progesterone may be coadministered.

Development

K5CreER;N1^fl;N2^fl;Ai9 mice (or K5-fl(N1/N2)-Ai9) are quadruple mutant animals assembled by Dr. James E. Schwob (Tufts University School of Medicine) that harbor the K5CreER^T2 transgene, the Notch1 floxed allele (N1^fl), the Notch2 floxed allele (N2^fl) and the R26R-LSL-tdTomato reporter allele (Ai9). Each allele/transgene is described below.

The Notch1 floxed allele (N1^fl ; Notch1^tm2Rko) was created in the laboratory of Dr. Raphael Kopan (while at Washington University School of Medicine) to have loxP sites flanking exon 1 of the notch1 locus (Notch1) on chromosome 2. Between 2011-2013, Dr. Schwob obtained N1^fl mice on an unspecified mixed genetic background from The Jackson Laboratory Stock No. 006951, and then used them as described below.

The Notch2 floxed allele (N2^fl ; Notch2^tm3Grid) was created by Dr. Thomas Gridley (while at The Jackson Laboratory) to have loxP sites flanking exon 3 of the notch2 locus (Notch2) on chromosome 3. Between 2011-2013, Dr. Schwob obtained C57BL/6-congenic N2^fl mice (black coat color) from The Jackson Laboratory Stock No. 010525, and then used them as described below.

The R26R-LSL-tdTomato reporter allele (Ai9; Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}) was created in the laboratory of Dr. Hongkui Zeng (Allen Institute for Brain Science) to have a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven tdTomato fluorescent protein; all inserted into the Gt(ROSA)26Sor locus on chromosome 6. Between 2008-2010, Dr. Schwob obtained C57BL/6J-congenic Ai9 mice (black coat color) from The Jackson Laboratory Stock No. 007909, and then used them as described below.

The K5CreER^T2 transgene (Tg(KRT5-cre/ERT2)2Ipc) was created in the laboratory of Dr. Pierre Chambon (IGBMC/GIE-CERBM) to have bovine keratin 5 promoter sequences (KRT5), a rabbit beta-globin intron II sequence, a CreER^T2 fusion gene (Cre-ER^T2; Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain) and an SV40 polyadenylation sequence. The ~8 kbp K5-Cre-ER^T2 transgene was microinjected into fertilized C57BL/6 x SJL F1 oocytes, and offspring from founder line 2 were established (see Stock No. 018394). Before 2009, Dr. Schwob obtained K5CreER^T2 mice on a C57BL/6;SJL genetic background (agouti coat color) from Dr. Randall Reed (Johns Hopkins University), and then used them as described below. To date (September 2017), the transgene copy number or insertion site is not known.

To generate the K5CreER;N1^fl;N2^fl;Ai9 quadruple mutant colony, Dr. Schwob bred C57BL/6J-Ai9 mice with C57BL/6;SJL-K5CreER^T2 mice. The bigenic animals were bred together and/or to C57BL/6J for multiple generations. Next, bigenic mice were bred to N1^fl and also to C57BL/6 N2^fl - each resulting in trigenic lines that were bred together to create the quadruple mutant colony. The donating investigator reports that the quadruple mutant colony was maintained on a predominantly C57BL/6 genetic background (showing black, agouti and occasionally white coat colors) as homozygous for the three targeted mutations and hemizygous for the K5CreER^T2 transgene. In 2017, quadruple mutant males were sent to The Jackson Laboratory as Stock No. 030855. Upon arrival, they were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish our live colony.

Allele

Symbol: Tg(KRT5-cre/ERT2)2Ipc
Name: transgene insertion 2, I Pierre Chambon
MGI accession ID: MGI:2177429
Generation method: Transgenic
Attributes: Recombinase-expressing; Inducible
Synonyms: CreERT2; K5-Cre-ER^T2; K5-CreERT2; Krt5^CreERT2; Tg(Krt1-5-cre/ERT2)2Ipc
Marker symbol: Tg(KRT5-cre/ERT2)2Ipc
Marker name: transgene insertion 2, I Pierre Chambon
Marker synonyms: CreERT2; K5-Cre-ER^T2; K5-CreERT2; Tg(Krt1-5-creERT2)2Ipc
Chromosome: UN
Strain of origin: (C57BL/6 x SJL)F1
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: Cre-ERT2 protein is expressed in epithelial cells.
Molecular note: This transgene expresses a tamoxifen-inducible form of cre recombinase under the control of the bovine KRT5 promoter in epidermal tissues.

Allele

Symbol: Notch2^tm3Grid
Name: targeted mutation 3, Tom Gridley
MGI accession ID: MGI:3617328
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Synonyms: N2^f; N2^flox; Notch2^f; Notch2^Fl; Notch2^flox
Marker symbol: Notch2
Marker name: notch 2
Marker synonyms: AGS2; HJCYS; hN2; Motch B; N2
Chromosome: 3
Strain of origin: 129S1/Sv-Oca2⁺ Tyr⁺ Kitl⁺
Molecular note: loxP sites were inserted to flank exon 3.

Allele

Symbol: Notch1^tm2Rko
Name: targeted mutation 2, Raphael Kopan
MGI accession ID: MGI:3044585
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Synonyms: fN1; N1^f; NICD1^fl; Notch1^f; Notch1^flox; Notch1^tm1Shn
Marker symbol: Notch1
Marker name: notch 1
Marker synonyms: 9930111A19Rik; AOS5; AOVD1; hN1; lin-12; Mis6; Motch A; N1; NOTCH; TAN1
Chromosome: 2
Strain of origin: 129X1/SvJ
Molecular note: LoxP were inserted flanking the first coding exon of the gene. An adjacent loxP flanked neomycin cassette was removed by Cre-mediated recombination in ES cells prior to production of chimeric animals.

Allele

Symbol: Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}
Name: targeted mutation 9, Hongkui Zeng
MGI accession ID: MGI:3809523
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: Ai9; Ai9(RCL-tdT; R26^LSL-tdTomato; ROSA:lxp-stp-lxp:tdTomato; Rosa26^Tomato; Rosa-CAG-LSL-tdTomato-WPRE; Rosa-LSL-TdTomato; Rs-tdTomato-R; Rosa26^LSL-tdTomato; tdTomato^LSL
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.
General note: This allele was used to generate Gt(ROSA)26Sor^{em1(CAG-tdTomato)Jahe} and Gt(ROSA)26Sor^{em2(CAG-tdTomato)Jahe}.