A CK19^CreERT knockin mouse line allows for conditional DNA recombination in epithelial cells in multiple endodermal organs

Abstract

SummaryCre/LoxP-mediated DNA recombination allows for gene function and cell lineage analyses during embryonic development and tissue regeneration. Here, we describe the derivation of a K19 CreERT mouse line in which the tamoxifen-activable CreER T was knocked into the endogenous cytokeratin 19 locus. In the absence of tamoxifen, leaky Cre activity could be detected only in less than 1% of stomach and intestinal epithelial cells, but not in pancreatic or hepatic epithelial tissues. Tamoxifen administration in postnatal animals induced widespread DNA recombination in epithelial cells of pancreatic ducts, hepatic ducts, stomach, and intestine in a dose-dependent manner. Significantly, we found that Cre activity could be induced in the putative gut stem/ progenitor cells that sustained long-term gut epithelial expression of a Cre reporter. This mouse line should therefore provide a valuable reagent for manipulating gene activity and for cell lineage marking in multiorgans during normal tissue homeostasis and regeneration.Keywords lineage tracing; pancreas; small intestine; colon; liver; kidney; stomach; Cre The Cre/LoxP-based technology allows for functional analyses of essential genes in specific organs by gene inactivation or controlled ectopic gene expression (Branda and Dymecki, 2004;Lewandoski, 2001;Sauer and Henderson, 1988). When combined with detectable marker protein expression, Cre-LoxP allows for cell lineage analyses in living animals (Branda and Dymecki, 2004;Gu et al., 2003). Upon modifying Cre to produce a tamoxifen (TM)-dependent molecule, CreER T , it is now possible to control Cre activity both spatially and temporally (Metzger and Chambon, 2001). This feature allows for dissecting the genetic requirements for cell/tissue homeostasis and for following cell lineages during tissue regeneration.We have derived a K19 CreERT knockin allele to recombine DNA in epithelial cells of several adult organs. K19 encodes an intermediate filament protein (Moll et al., 1982) that is expressed in multiple cell types from the epiblast stage and is maintained in multiple epithelial cell types of later embryonic and postnatal stages (Bosch et al., 1988;Lane et al., 1983;Moll et al., 1982;Quinlan et al., 1985). For example, K19 is highly expressed in the pancreatic ducts of the adult pancreas (Deramaudt et al., 2006), but is absent or weak in acini and islets (Brembeck et al., 2001 We derived a K19 CreERT allele by replacing K19 ATG with a CreER T -cDNA followed by a SV40 polyadenylation signal (see Fig. 1). This design minimally altered K19 transcription regulatory elements while producing a CreER T message with a short 3′-UTR. Inclusion of a polyadenylation signal 3′ to the CreER T sequence prevented the transcription of the five noncoding exons of the endogenous K19 gene. Otherwise, the presence of these noncoding exons in CreER T mRNA could trigger nonsense-mediated mRNA degradation (Conti and Izaurralde, 2005;Doma and Parker, 2007). Thus, adding an extra polyadenylation signal immediately down-stream of CreER T c...

Identifying the cellular origin of squamous skin tumors

“…We first induced the expression of KRas G12D in bulge SCs and their progeny using mice expressing inducible CRE under the regulatory region of HF-specific promoter [K15-CREPR (12) and K19CREER (30)]. We did observe some leakiness in the K15CREPR/RosaYFP mice, visible by the presence of patches of YFP-expressing cells in the absence of RU486 (Fig.…”

Hair follicle and interfollicular epidermal stem cells make varying contributions to wound regeneration

“…25 When coupled with the LacZ reporter allele, K19;LacZ mice displayed fewer labeled bulge cells compared to K15-Cre PR1 mice ( Figure S2); however, labeling was more specific to the bulge than in K15-Cre PR1 animals, since labeled non-bulge cells were rarely observed in K19;LacZ mice. When we wounded these animals, labeled cells were also retained in regenerated skin up to 50 days after injury (Fig.…”

“…The following strains were used: Tg(KRT14-cre/ERT)20Efu (K14-Cre ERT ) 21 ; Tg(Krt-15-cre/PGR)22Cot (K15-Cre PR1 ) 20 ; Shhtm1(EGFP/cre)Cjt (Shh-Cre) 43 ; Krt19 tm1(cre/ERT)Ggu (K19-Cre ERT ) 25 ; Gt(ROSA)26Sortm1(EYFP)Cos 44 ; Gt(ROSA) 26Sortm1Sor 45 ; and ROSA26A-dnMAML1-GFP (dnMAML). 28 All animals contained single copies of the transgene and reporter alleles.…”

Loss of liver E-cadherin induces sclerosing cholangitis and promotes carcinogenesis

“…K19 CreERT mice were a generous gift from Guoqiang Gu (Vanderbilt University, Nashville, TN) (18). All mice were on the C57BL/6 genetic background.…”

“…2B). Next, to delete E-cadherin only in BECs, we crossed CDH1 F/F mice with K19 CreERT mice in which tamoxifen (TAM)-inducible Cre ERT was knocked into the endogenous K19 locus (CDH1 F/F /K19 CreERT ) (18). According to the previous study, which showed relatively low efficacy of recombination in BECs, we injected TAM twice, at 5 and 9 wk after birth.…”