Rosa26loxp stop loxp tdtomato

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The ROSA26loxP-STOP-loxP-tdTomato is a genetic construct that includes the ROSA26 locus, a loxP-flanked STOP cassette, and the tdTomato fluorescent protein gene. This construct allows for Cre-mediated, conditional expression of the tdTomato reporter.

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11 protocols using rosa26loxp stop loxp tdtomato

Genetic Mouse Models for Auditory Research

Cited 3 times

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Sox9-CreER^T2 (stock #18829, Kopp et al., 2011 (link)), Rosa26^{loxP-stop-loxP-tdTomato} (Rosa26^tdTomato, also called Ai14, stock #7914, Madisen et al., 2010 (link)), Atoh1^loxP/loxP (stock #8681; Shroyer et al., 2007 (link)), and Atoh1^GFP (stock #13593, Rose et al., 2009 (link)) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Pou4f3^DTR (DTR) mice (Golub et al., 2012 (link); Tong et al., 2015 (link)), a gift from Ed Rubel at the University of Washington, were used as heterozygotes. DTR:Atoh1^GFP/GFP mice were on a C57BL/6J background. DTR:Sox9-CreER^T2:Rosa26^tdTomato:Atoh1^loxP/loxP (Atoh1 CKO) and DTR:Sox9-CreER^T2:Rosa26^tdTomato:Atoh1^+/+ (Atoh1 WT) were on a C57BL/6J:BALBc:CD1 background. Both genders were used in all studies.
All animal procedures were conducted at the Southern Illinois University School of Medicine (Springfield, IL) using approved protocols from their animal care and use committees. Animal numbers are provided in Table 1. The “n” value represents the number of mice included in each experiment.

Hicks K.L., Wisner S.R., Cox B.C, & Stone J.S. (2019). Atoh1 is required in supporting cells for regeneration of vestibular hair cells in adult mice. Hearing research, 385, 107838.

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Targeted Transgenic Mouse Generation

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The targeting constructs were built using the W vector (provided by J. Segal and K. Rajewsky, Immune Disease Institute, Boston). Homologous arms were cloned by PCR from genomic DNA of C57BL/6 mice. Constructs were sequenced validated. ES cells (B6/3) derived from C57BL/6 mice were electroporated with linearized constructs and clones were positively selected with G418 and negatively selected by the diphtheria toxin cassette of the constructs. Clones were manually picked, expanded, and screened by Southern blot. Positive clones were expanded, validated, and injected into blastocysts, and germline transmission was confirmed. All strains used were on a C57BL/6 background including CD45.1 and CD45.2 congenic, and the Rosa26-LoxP-STOP-LoxP-tdTomato (stock# 007914) was obtained from The Jackson Laboratory. All experiments involving mice were done per institutional guidelines of The Harvard Medical School Standing Committee on Animals, with IACUC approval.

Gazit R., Mandal P.K., Ebina W., Ben-Zvi A., Nombela-Arrieta C., Silberstein L.E, & Rossi D.J. (2014). Fgd5 identifies hematopoietic stem cells in the murine bone marrow. The Journal of Experimental Medicine, 211(7), 1315-1331.

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Transgenic Mouse Lines for Lineage Tracing

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C57BL/6J and transgenic mouse lines were used, including Lhx6-Cre^ER (JAX#010776, The Jackson Laboratory) [28 (link)], ROSA26^{loxP-STOP-loxP-tdTomato} (tdTomato conditional reporter, JAX#007905, The Jackson Laboratory) [55 (link)], Gli1-LacZ (JAX#008211, The Jackson Laboratory) [56 (link)], Gli1-Cre^ER (JAX#007913, The Jackson Laboratory) [57 (link)] and Ctnnb1^floxE3 [58 (link)]. All mice were housed in pathogen-free conditions, and newborn pups were documented as postnatal stage PN0.5. Both male and female mice at indicated stages were collected, genotyped and analyzed.

He J., Jing J., Feng J., Han X., Yuan Y., Guo T., Pei F., Ma Y., Cho C., Ho T.V, & Chai Y. (2021). Lhx6 regulates canonical Wnt signaling to control the fate of mesenchymal progenitor cells during mouse molar root patterning. PLoS Genetics, 17(2), e1009320.

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Transgenic Mouse Strains for Lineage Tracing

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The following mouse strains were purchased from the Jackson Laboratory: Rosa26^CreERT2 (B6;129-Gt(ROSA)26Sortm1(cre/ERT)Nat/J, RRID: IMSR_JAX:004847); Lgr5^{EGFP-iREs-creERT2} (B6.129P2-Lgr5tm1(cre/ERT2)Cle/J, RRID :IMSR_JAX:008875); Rosa26^{LoxP-Stop-LoxP-tdTomato} (B6.Cg-Gt(ROSA)26Sortm14 (CAG-tdTomato) Hze/J, RRID: IMSR_JAX:007914); and p53^loxP/loxP (B6.129P2-Trp53tm1Brn/J, RRID: IMSR_JAX:008462). The Prdm16 conditional knockout mouse line was generated by Patrick Seale and Bruce Spiegelman and is available from the Jackson Laboratory: Prdm16^loxP/loxP (B6.129-Prdm16tm1.1Brsp/J, RRID: IMSR_JAX:024992). All animal work was approved by the University of Pennsylvania’s Institutional Animal Care and Use Committee. Mice were housed under the care of University of Pennsylvania University Laboratory Animal Resources (ULAR), which provides both basic husbandry and veterinary care. Animals were specific-pathogen free (SPF) and raised at room temperature on standard chow with a 12-hour light/dark cycle. Both sexes were used for experiments. All experiments were done on adult animals between the ages of 6 to 10 weeks at the onset of the experiment.

Stine R.R., Sakers A.P., TeSlaa T., Kissig M., Stine Z.E., Kwon C.W., Cheng L., Lim H.W., Kaestner K.H., Rabinowitz J.D, & Seale P. (2019). PRDM16 maintains homeostasis of the intestinal epithelium by controlling region-specific metabolism. Cell stem cell, 25(6), 830-845.e8.

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Genetic Manipulation of Mouse Models

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Gli1-Cre^ERT2 mice were from L. Baojie (59 (link)); Alk3^fl/fl mice were provided by Y. Mishina (60 (link)); Smad4^fl/fl mice were provided by Y. Xiao (61 (link)); Rosa26-Loxp-STOP-Loxp-tdTomato mice were obtained from the Jackson Laboratory; Muc2-mCherry mice and Apoa1-mCherry mice were commercially generated by Gem Pharmatech (Nanjing). Both male and female mice ranging from 2 to 4 months old in age were used. No statistical method was used to predetermine sample size. In general, we used at least three mice per genotype in each experiment. For Cre induction, mice were intraperitoneally injected with 100 μl of tamoxifen dissolved in oil at 20 mg/ml for five consecutive days. All animal studies were performed in accordance with the guidelines and under the approval of the Institutional Animal Care and Use Committee of Tsinghua University (YGC-19).

Wang Y., Lou R., Zhang Z., Xiao C., Yu S., Wei S., Liu Y., Fu W., Li B, & Chen Y.G. (2023). Stromal BMP signaling regulates mucin production in the large intestine via interleukin-1/17. Science Advances, 9(43), eadi1827.

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Generating Pax9-CreER;tdTomato Reporter Mice

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The Pax9-CreER founder mouse (F0) was mated with Bl6 females to generate F1 Pax9-CreER mice. ROSA26LoxP−STOP−LoxP−tdTomato (tdTomato reporter) mice were obtained from the Jackson Laboratory (JAX no. 007905) (Madisen et al., 2010 (link)). tdTomato reporter female mice were mated with Pax9-CreER F1 male mice and received 1.5mg/10g bodyweight tamoxifen (Sigma T5648) at E8.5 through E14.5. The female mice were euthanized 48 hours after injection to collect Pax9-CreER;tdTomato embryos. Adult Pax9-CreER;tdTomato mice received 5 daily injections of 1.5 mg/10 g bodyweight tamoxifen and were euthanized 48 hours after the last injection. Whole samples were imaged using a Leica MZ10F. For cryosections, samples at various stages were fixed in 4% paraformaldehyde, passed through a sucrose series, then embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura) and frozen onto a dry ice block to solidify. Embedded samples were cryosectioned at 7μm thickness using a cryostat (Leica CM1850). Sections were counterstained with DAPI (Sigma, D9542). Images were captured using a fluorescence microscope (Leica DMI 3000B) with filter settings for DAPI/FITC/TRITC.

Feng J., Jing J., Sanchez-Lara P.A., Bootwalla M.S., Buckley J., Wu N., Yan Y, & Chai Y. (2016). Generation and Characterization of Tamoxifen-Inducible Pax9-CreER Knock-In Mice using CrispR/Cas9. Genesis (New York, N.Y. : 2000), 54(9), 490-496.

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Genetic Mouse Models for Ror2 Study

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Osr2-Cre mice were a gift from Rulang Jiang (Cincinnati Children's Hospital, OH, USA) (Lan et al., 2007 (link)). K14-rtTA (007678; Xie et al., 1999 (link)), Teto-Cre (006234; Perl et al., 2002 (link)), Ror2^fl/fl (018354; Ho et al., 2012 (link)), and ROSA26^{loxP-STOP-loxP-tdTomato} (tdTomato conditional reporter, 007905; Madisen et al., 2010 (link)) mouse lines were purchased from The Jackson Laboratory. Osr2-Cre;Ror2^fl/+ male mice were crossed with Ror2^fl/fl female mice to generate Osr2-Cre;Ror2^fl/fl mice. K14-rtTA;Teto-Cre;Ror2^fl/fl male mice were crossed with K14-rtTA;Ror2^fl/fl female mice to generate K14-rtTA;Teto-Cre;Ror2^fl/fl mice. A doxycycline rodent diet was administered every day from E14.5. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Southern California (USC).

Ma Y., Jing J., Feng J., Yuan Y., Wen Q., Han X., He J., Chen S., Ho T.V, & Chai Y. (2021). Ror2-mediated non-canonical Wnt signaling regulates Cdc42 and cell proliferation during tooth root development. Development (Cambridge, England), 148(2), dev196360.

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Conditional Knockout Mice for Epigenetic Studies

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All experiments according to IACUC-approved protocols. Dnmt1^fl/fl (Fan et al., 2001 (link); Jackson-Grusby et al., 2001 (link)) and Dnmt3a^fl/fl (Kaneda et al., 2004 (link)) mice on a C57BL/6 background were crossed with Olig1-cre (Jackson Laboratory) or Cnp-cre mice (Lappe-Siefke et al., 2003 (link)) or Rosa26-loxP-STOP-loxP-TdTomato (Jackson Laboratory).

Moyon S., Huynh J.L., Dutta D., Zhang F., Ma D., Yoo S., Lawrence R., Wegner M., John G.R., Emery B., Lubetzki C., Franklin R.J., Fan G., Zhu J., Dupree J.L, & Casaccia P. (2016). Functional characterization of DNA methylation in the oligodendrocyte lineage. Cell reports, 15(4), 748-760.

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Genetic Mouse Models of Bone and Cancer

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Col2a1-creERT2,⁸ Osx-creERT2,^{3 (link)} Col1-creERT2,^{9 (link)} and Cre-dependent Kras transgenic, loxP-stop-loxP-Kras^G12D^{10 (link)} mice have been described elsewhere. The Cre reporter, Rosa26-loxP-stop-loxP-tdTomato mice were acquired from Jackson Laboratory (Bar Harbor, ME, USA).^{7 (link)} All procedures were conducted in compliance with the Guideline for the Care and Use of Laboratory Animals approved by Massachusetts General Hospital's Institutional Animal Care and Use Committee (IACUC). Mice were in a mixed genetic background. Comparisons were made between littermates. Tamoxifen injections were given i.p. at a dose of 0.1 mg/g to pregnant females as described elsewhere.^{4 (link)}

Papaioannou G., Mirzamohammadi F, & Kobayashi T. (2016). Ras signaling regulates osteoprogenitor cell proliferation and bone formation. Cell Death & Disease, 7(10), e2405-.

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Genetically Engineered Mouse Lines for Developmental Biology

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All mice were housed in specific pathogen-free, Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC)-approved facilities at the Columbia University Irving Medical Center. Food and water were provided ad libitum over 12-hour light/dark cycles. Lepr-Cre (JAX stock #032457)^{66 (link)}, Gli1-CreER (JAX stock #007913)^{66 (link)}, Gli1-LacZ (JAX stock #008211)^{27 (link)}, Rosa26-loxp-stop-loxp-tdTomato (Ai9, JAX stock #007909)^{67 (link)}, Rosa26-loxp-stop-loxp-iDTR (JAX stock #007900)^{68 (link)}, Pdgfra-H2B-GFP (JAX stock #007669)^{24 (link)}, and Sox10-CreER (JAX stock #027651)^{46 (link)} mice were obtained from the Jackson Laboratory. Col1a1(HS4,5–3.2 kb)-GFP^{25 (link)} and Pdgfra-DreER^{26 (link)} were described previously. Rosa26-rox-stop-rox-eGFP (RC∷RG) mice were generated by crossing RC∷RLTG mice (JAX stock #026931)^{69 (link)} with E2a-Cre mice (JAX stock #003724)^{69 (link)} to induce germline removal of the loxp-flanked tdTomato cassette from the Rosa26 locus. Young adult mice (both males and females of 6–12 weeks old) were used in this study. All experimental protocols were approved by Columbia University’s Institutional Animal Care and Use Committee.

Moores S.L., Sabry J.H, & Spudich J.A. (1996). Myosin dynamics in live Dictyostelium cells.. Proceedings of the National Academy of Sciences of the United States of America, 93(1), 443-446.

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