huLangerin-Cre, huLangerin-CreERT2, TGF-βloxP and TGF-βRI–CA and huLangerin-DTA mice have been previously described17 (link),26 (link),44 (link). huLangerin-CreERT2 mice were crossed to the TGF-βRI–CA line to generate TGF-βRI–CALC mice. Itgb6−/− and Itgb8loxP mice were provided by D. Sheppard (University of California, San Francisco). C57BL/6 (wild-type) and K14-Cre mice were purchased from Jackson Laboratories. K14-Cre and huLangerin-Cre mice were crossed with Itgb8loxP mice to obtain Itgb8ΔKC mice and Itgb8ΔLC mice, respectively. We used age-matched female mice that were between 4 and 12 weeks of age in all our experiments. All mice were housed and bred in microisolator cages and were fed irradiated food and acidified water. The University of Minnesota Institutional Care and Use Committee approved all the experimental procedures on mice.
K14 cre mice
Manufactured by Jackson ImmunoResearch
Sourced in United States, Montenegro
K14-Cre mice are a transgenic mouse strain that expresses the Cre recombinase enzyme under the control of the keratin 14 (K14) promoter. The K14 promoter directs Cre expression in the basal layer of stratified squamous epithelia, including the skin, oral mucosa, esophagus, and cornea.
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12 protocols using k14 cre mice
1
Genetic Mouse Models for Studying TGF-β Signaling
2
Generation of Runx2 Conditional Knockout Mice
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K14-Cre mice (004782; the Jackson Laboratory), which express Cre recombinase in the oral mucosal epithelium at approximately 10.5 days post-coitum, were mated with Runx2flox/flox mice to create K14-Cre+;Runx2flox/wt mice. The K14-Cre+;Runx2flox/wt mice were then bred with Runx2flox/flox mice to generate K14-Cre+;Runx2flox/flox (cKO) mice. DNA samples from mouse tails were genotyped by PCR. PCR primers as a, b, c and d described above (Supplementary Fig. 1a ) were used to identify Runx2flox/flox mice from the offspring. In addition, a set of specific primers for the Cre transgene was adopted to identify Cre recombinase, forward, 5′-TTCCTC AGGAGTGTCTTCGC-3′ (“e” in Supplementary Fig. 1a ); reverse, 5′- GTCCATGTCCTTCCTGAAGC-3′ (“f” in Supplementary Fig. 1a ). PCR with the Cre-specific primers yielded a 494-bp fragment from mice expressing Cre recombinase, and no PCR product was amplified from mice expressing the (wild type) WT allele (Supplementary Fig. 1d ). The mice were maintained in a conventional animal care facility and given free access to standard mouse chow and water. Animals were sacrificed by CO2 asphyxiation.
3
Conditional Knockout of Ift140 in Stratified Squamous Epithelial Cells
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The mice used in this study were maintained on a C57/Bl6 background. Ift140flox/fox mice were produced as previously described.17 (link) The primers for genotyping included: Forward, 5′-ATCTTAATTTGTGTTGAAGGGGTT-3′, and Reverse, 5′-CTGCCAGGGGTACATGGTAGTAAG-3′. They were crossed with K14-Cre mice (purchased from the Jackson Laboratory) to generate K14+ SSPC-selective Ift140-cKO mice. The genotyping method of the Ift140flox/fox and Ift140-cKO mice were presented in Appendix Fig. S1 . For the genetic linage trancing study, Ift140-CreERT2 mice were generated as previously reported.16 The identification of Ift140-CreERT2 mice included the forward primer, 5′-CTAATGGTTCCAGTCTGCAGGCCC-3′, and the reverse primer, 5′-CAATAAGACCAGGCACATACCATC-3′. Then, Ift140-CreERT2 mice were crossed with the reporter strain Rosa26tdTomato (purchased from Cyagen Biotechnology). Mice were raised in a specific pathogen-free facility under a 12:12-h day/night illumination cycle. All procedures and protocols were approved by The Animal Welfare Committee of Tongji University, Hospital of Stomatology (2019-DW-004).
4
Genetic Mouse Models for Cancer Research
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Animal work was carried out with ethical approval from University of Glasgow under the revised Animal (Scientific Procedures) Act 1986 and the EU Directive 2010/63/EU (PPL 60/4181). All experiments were performed in accordance with relevant guidelines and regulations. Animals were housed in individual ventilated cages in a barrier facility proactive in environmental enrichment. The generation and characterization of Runx2fl/fl conditional knock-out mice is described in detail in Supplementary Fig. S1 . K14-Cre mice22 (link) were obtained from The Jackson Laboratory (USA). The Z/EG, MMTV-PyMT, MMTV-Her2 and BLG-Cre/Brca1fl/fl/p53+/– mice have been described previously23 (link)38 (link)58 (link)59 (link). Apc1572T mouse tumour samples were kindly provided by Professor Riccardo Fodde. BLG-Cre/Ptenfl/fl/Apcfl/fl mouse tumour samples and the Catnb+/lox(ex3) mouse model were kindly provided by OJ Sansom. FVB and CD1-nude mice were obtained from Charles River Research Models & Services (UK).
5
Generation and Analysis of HVEM Knockout Mice
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6–8-wk-old male WT, LIGHT−/− (Scheu et al., 2002 (link)), and HVEMflox/flox mice crossed to K14-cre mice (Jackson Labs) were bred in-house on the C57BL/6 background. HVEMfl/fl mice were generated at the La Jolla Institute. A frt site-flanked LacZ reporter cassette was inserted into intron 2 of the Tnfrsf14 (HVEM) gene and a variant frt site (F3)-flanked Neor cassette, for drug selection, was inserted into intron 6 of the Tnfrsf14 gene by recombineering. Germline transmitted Tnfrsf14flox-neo/flox-neo mice were crossed with mice expressing the FLPe variant of the Saccharomyces cerevisiae FLP1 recombinase mice that deleted the LacZ reporter and the Neo cassette to generate HVEMflox/flox mice. In some experiments, WT BALB/c and IL-4R alpha-deficient mice (Jackson Labs) were used. All experiments were in compliance with the regulations of the La Jolla Institute for Allergy and Immunology Animal Care Committee.
6
Genotyping PRMT1 Knockout Mice
7
Genetically Engineered Mice for Research
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C57/B6 mice (8-week-old) were purchased from the Experimental Animal Center of the Kunming Institute of Zoology (China). The Mcam floxed mice (8-week-old) have been described previously30 (link). The MMTV-Cre (line D) and K14-Cre mice (8-week-old) were obtained from the Jackson Laboratory (USA). The female NOD/SCID mice (3-week-old) were purchased from the company of Charles River. The mice were raised in a specific pathogen free (SPF) environment with an ambient temperature of 18-22 °C, a humidity of 50%-60%, and a 12 h light-dark cycle. All experimental procedures and animal care and handling were performed according to the protocols approved by the Ethics Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences (IACUC-RE-2023-07-006).
8
Keratinocyte-Specific HDAC8/9 Knockout Mice
9
Generating caACVR1;K14-Cre Mice
10
Murine Models for Skin Cancer
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All mice were housed in the Animal Care Facility at the University of Texas Southwestern Medical Center, and all procedures were approved by Institutional Animal Care and Use Committee (IACUC) at the University of Texas Southwestern Medical Center and conformed to NIH guidelines. All mice strains were maintained in a room with 12/12 (day/night) light cycle with a temperature of 70–72 °F. K14Cre mice were available from the Jackson Laboratory (Bar Harbor, ME). All mice were maintained on a mix C57bl/6 background. DMBA/TPA treatment was performed according to Abel et al.22 (link). using a single dose of DMBA (25 μg) and a bi-weekly schedule for TPA (4 μg) over 23 weeks. The PLPCreERT2; Nf1f/−; ROSA-LacZ mice31 (link) was conditionally activated with a single subcutaneous dose of 4-hydroxytamoxifen (40 μg) dissolved in 100% ethanol on the first postnatal day. Genotyping was performed by PCR as reported elsewhere14 (link),23 (link),26 (link),67 (link). A total number of 59 mice (DMBA/TPA study) and 216 mice (MPNST study) were used in this report. For the DMBA/TPA study, full randomization was not possible to maximize the number of same sex mice and enrolled on treatment or vehicle at 3 months old. The investigators were not blinded to the group allocation during the experiment. No mice were excluded for any reasons.
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