Villin creert2

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Villin-CreERT2 is a transgenic mouse line that expresses a tamoxifen-inducible Cre recombinase under the control of the villin promoter. The Villin-CreERT2 mouse allows for the inducible Cre-mediated recombination of loxP-flanked target genes specifically in intestinal epithelial cells.

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5 protocols using villin creert2

Genetic Mouse Models for Intestinal Research

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Animal experiments were approved by the National Animal Experiment Board of Finland. Mice were housed and monitored according to the Federation of European Laboratory Animal Science Associations guidelines and recommendations. The mice were weighed and their health was closely monitored during the experiment. The mouse lines Lef1^fl/fl (25 (link)), Apc^fl/fl (76 (link)), Lgr5-EGFP-IRES-Cre^ERT2 (4 (link)), Rosa26^LSL-TdTomato (Jackson Laboratory, stock no. 021875), Apc^Min/+ (Jackson Laboratory, stock no. 002020), Villin-Cre^ERT2 (Jackson Laboratory, stock no. 020282), and Villin-Cre (Jackson Laboratory, stock no. 021504) have been described previously. Lef1^fl/fl mice with mixed C57BL/6 and 129SV background were used after backcrossing to the C57BL/6 strain for >6 generations. All experiments were performed three times with independent cohorts. Approximately equal numbers of male and female mice of same age were used for all the experiments.

Heino S., Fang S., Lähde M., Högström J., Nassiri S., Campbell A., Flanagan D., Raven A., Hodder M., Nasreddin N., Xue H.H., Delorenzi M., Leedham S., Petrova T.V., Sansom O, & Alitalo K. (2021). Lef1 restricts ectopic crypt formation and tumor cell growth in intestinal adenomas. Science Advances, 7(47), eabj0512.

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Generating Inducible Transgenic Mouse Models

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All inducible Cre lines (R26R-CreERT2: JAX006965, Villin-CreERT2: JAX020282, Lgr5-EGFP-IRES-CreERT2: JAX008875, Sftpc-CreERT2: JAX028054, Krt5-CreERT2: JAX02915), the R26R-Confetti line (JAX017492), LSL-Kras^G12D (JAX008179) line, Apc^fl/fl line (JAX009045) and Apc^Min line (JAX002020) were obtained from The Jackson Laboratory. Pik3ca^Lat-H1047R line was donated by W.A Phillips^{41 (link)}. Red2Onco targeting vectors were generated by gene synthesis. Oncogene sequences were obtained from Addgene (Notch1ICD: Addgene plasmid #15079^{42 (link)}, Kras^G12D: Addgene plasmid #11549^{43 (link)}, PIK3CA^H1047R: Addgene plasmid #12524^{44 (link)}). Mice were created by inserting the Red2Onco cassette into the tdimer2 locus in ES cells obtained from R26R-Confetti mice using CRISPR-Cas9 nickase-mediated homologous recombination. Insertion of the oncogenic sequence was confirmed by long-range PCR. Specific genotyping primers were designed outside of the homology arms and were used in combination with primers within the knock-in cassette.

Yum M.K., Han S., Fink J., Wu S.H., Dabrowska C., Trendafilova T., Mustata R., Chatzeli L., Azzarelli R., Pshenichnaya I., Lee E., England F., Kim J.K., Stange D.E., Philpott A., Lee J.H., Koo B.K, & Simons B.D. (2021). Tracing oncogene-driven remodelling of the intestinal stem cell niche. Nature, 594(7863), 442-447.

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Generation of Conditional IL22 and MHCII Mice

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Il22^hCD4.fl reporter/floxed and Il22^∆Tcell cKO mice were previously generated within our laboratory^{4 (link)}. C57BL/6 (wild type), C3H/HeJ, H2-Ab1 floxed, Cd4-cre, SMARTA-1^{59 (link)} CD45.1, Tcra^–/–, Villin-cre and Villin-cre/ERT2 mice were purchased from Jackson Laboratory. H2-Ab1^Villin (H2-Ab1 floxed × Villin-cre) cKO mice were screened by PCR and flow cytometry to exclude mice with spontaneous, Cre-independent germline deletion of MHCII^{60 (link)}. In most experiments, littermates were used as controls and experimental adult animals (8–12 wk old) were co-caged in groups of 2–7 mice. Male and female mice were used whenever possible. All mouse strains were bred and maintained at University of Alabama at Birmingham (UAB) in accordance with IACUC guidelines.

Zindl C.L., Wilson C.G., Chadha A.S., Duck L.W., Cai B., Harbour S.N., Nagaoka-Kamata Y., Hatton R.D., Gao M., Figge D.A, & Weaver C.T. (2024). Distal colonocytes targeted by C. rodentium recruit T-cell help for barrier defence. Nature, 629(8012), 669-678.

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Generating Inducible Mouse KO Models

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Inducible global, intestine‐ and kidney tubule‐specific LAT4 KO mouse models were generated by breeding LAT4^f/f mice with mice containing Rosa26‐CreER^T2+ (B6.129‐Gt(ROSA)26Sortm1(cre/ERT2)Tyj/J) (IMSR catalogue no. JAX:008463, RRID:IMSR_JAX:008463; The Jackson Laboratory, Bar Harbor, ME, USA) (Ventura et al. 2007), Villin‐CreER^T2+ (B6N.Cg‐Tg(Vil1‐cre/ERT2)23Syr/J) (IMSR catalogue no. JAX:020282, RRID: IMSR_JAX:020282; The Jackson Laboratory) (El Marjou et al. 2004) and Pax8‐rtTA⁺/LC1⁺ (B6.Cg‐Tg(Pax8‐rtTA2S*M2)1Koes/J) (IMSR catalogue no. JAX:007176, RRID:IMSR_JAX:007176; The Jackson Laboratory) (Traykova‐Brauch et al. 2008) transgenes, respectively (Fig. 1). Constitutional intestine‐specific LAT4 KO mice were produced by breeding LAT4^f/f mice with Villin‐Cre⁺ mice [B6.Cg‐Tg(Vil1‐cre)997Gum/J] (IMSR catalogue no. JAX:004586, RRID:IMSR_JAX:004586; The Jackson Laboratory) (El Marjou et al. 2004). Transgenic Pax8‐rtTA⁺/LC1⁺ and Villin‐Cre⁺ mice were kind gifts from Dr N. Hernando, University of Zurich (Zurich, Switzerland).

Rajendran A., Poncet N., Oparija‐Rogenmozere L., Herzog B, & Verrey F. (2020). Tissue‐specific deletion of mouse basolateral uniporter LAT4 (Slc43a2) reveals its crucial role in small intestine and kidney amino acid transport. The Journal of Physiology, 598(22), 5109-5132.

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Conditional Genetic Manipulation of Intestine

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Animal handling and experimental procedures conformed to institutional guidelines and were approved by the Sanford-Burnham-Prebys Medical Discovery Institute Institutional Animal Care and Use Committee, and by the Weill Cornell Medicine Institutional Animal Care and Use Committee. Prkci^f/f (Leitges et al., 2001 (link); Nakanishi et al., 2016 (link)), Villin-Cre (Madison et al., 2002 (link)), and Villin-CreERT2 (el Marjou et al., 2004 (link)) mice were previously described. Prkci^f/f;Villin-Cre and Prkci^f/f;Villin-CreERT2 mice were generated by breeding Prkci^f/f mice with Villin-Cre mice (Jackson Laboratory, stock number 012641) or Villin-CreERT2 (Jackson Laboratory, stock number 020282) mice respectively. All mouse strains were generated in a C57BL/6 background and were born and maintained under pathogen-free conditions. All mice were maintained on food and water ad libitum and were age-matched and co-housed for all experiments. Mice were sacrificed and small intestine, colon or other organs were collected for analysis. All genotyping was done by PCR. Age- and sex-matched mice were used for all experiments.

Linares J.F., Zhang X., Martinez-Ordoñez A., Duran A., Kinoshita H., Kasashima H., Nakanishi N., Nakanishi Y., Carelli R., Cappelli L., Arias E., Yashiro M., Ohira M., Patel S., Inghirami G., Loda M., Cuervo A.M., Diaz-Meco M.T, & Moscat J. (2021). PKCλ/ι inhibition activates an ULK2-mediated interferon response to repress tumorigenesis. Molecular cell, 81(21), 4509-4526.e10.

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