Cre-mediated recombination in the skin melanocyte lineage

Organ-specific expression of a Cre recombinase allows the analysis of gene function in a particular tissue or cell type. Using a 6.1 kb promoter from the mouse tyrosinase gene, we generated and characterized two lines of transgenic mice that express Cre recombinase in melanoblasts. Utilizing a Cre-responsive reporter mouse strain, genetic recombination was detected in the melanoblasts of the skin from embryonic day 11.5. In addition, Cre-expression was detected in the skin and eyes of mice. Cre transgene activity was occasionally detected in the brain and peripheral nerves but not in other tissues. When Tyr::Cre mice were crossed with mice carrying a homozygous loxP conditional mutation for the insulin-like growth factor receptor gene (Igf1r), Cre-melanoblast-specific recombination pattern was confirmed and no abnormal phenotype was observed. In conclusion, Tyr::Cre transgenic mice provide a valuable tool to follow the cell lineage and to examine gene function in melanocyte development and transformation.     

Efficient inducible Cre-mediated recombination in Tcf21 cell lineages in the heart and kidney

Tcf21 is a Class II bHLH family member with essential roles in the formation of the lungs, kidneys, gonads, spleen, and heart. Here, we report the utility of a mouse line with targeted insertion of a tamoxifen-inducible Cre recombinase, MerCreMer at the Tcf21 locus. This mouse line will permit the inducible expression of Cre recombinase in Tcf21-expressing cells. Using ROSA26 reporter mice, we show that Cre recombinase is specifically and robustly activated in multiple Tcf21-expressing tissues during embryonic and postnatal development. The expression profile in the kidney is particularly dynamic with the ability to cause recombination in mesangial cells at one time of induction and podocytes at another time. These features make the Tcf21-driven inducible Cre line (Tcf21(iCre) ) a valuable genetic tool for spatiotemporal gene function analysis and lineage tracing of cells in the heart, kidney, cranial muscle, and gonads.     

Temporal Cre-mediated recombination exclusively in endothelial cells using Tie2 regulatory elements

Summary: The versatility of the bacteriophage Cre/LoxP system is dependent on the availability of a spectrum of tissue-specific Cre transgenic mice to address a host of biological questions. In this paper, we report on the generation of an inducible Tie2Cre transgenic mouse line that facilitates gene targeting exclusively in endothelial cells. The temporal manner of recombination is feasible through the use of a Cre-estrogen receptor fusion protein ER(T2) and was, in practical terms, achieved by feeding the animals the estrogen antagonist tamoxifen orally for 5 weeks. High efficiency of recombination was found in the vast majority of endothelial cell populations examined, as monitored by an EGFP reporter mouse line. Critically, no EGFP expression was observed in any uninduced mice. This inducible Cre line will be a very beneficial asset to investigating the role of endothelial specific genes in the adult mouse and to induce transgenes in the endothelium in an extremely efficient manner. genesis 33:191-197, 2002.     

Targeted insertion of an IRES Cre into the Hnf4alpha locus: Cre-mediated recombination in the liver, kidney, and gut epithelium

The Hnf4alpha gene belongs to a family of trancriptional regulators required for liver development and function. Hnf4alpha is also expressed in other tissues, including the newly formed visceral endoderm of the early postimplantation embryo, and later in embryogenesis in the gut epithelium and the kidney. The regulatory sequences involved in controlling expression of Hnf4alpha at these diverse sites are not clearly understood. Here we used homologous recombination to introduce Cre recombinase coding sequences into the endogenous Hnf4alpha locus. Crossing Hnf4alpha(Creex2/+) mice with R26R partners allowed us to follow the pattern of Cre-mediated recombination. Our results show that recombination of the reporter allele closely follows endogenous Hnf4alpha expression, but with a slight temporal delay. Thus, the Hnf4alpha(Creex2) strain should prove useful for conditionally deleting gene activity in the liver, gut epithelium, or kidney.     

Tamoxifen‐inducible Cre‐mediated recombination in adipocytes

To generate a mouse line which allows inducible, Cre/loxP‐dependent recombination in adipocytes, we used RedE/RedT‐mediated recombineering to insert the CreER^T2‐transgene, which encodes a fusion protein of Cre and a mutated tamoxifen‐responsive estrogen receptor, into the start codon of the adipocyte‐specific Adipoq gene. Adipoq encodes adiponectin, an adipokine specifically expressed in differentiated adipocytes. Tamoxifen treatment induced almost complete recombination in white adipose tissue of the AdipoqCreER^T2 mouse line (97%–99%), while no recombination was seen in vehicle‐treated animals. Recombination in brown adipose tissue was about 15%, whereas other organs and tissues did not undergo recombination. In addition, mice expressing CreER^T2 in adipocytes did not show any alterations of metabolic functions like glucose tolerance, lipolysis, or energy expenditure compared to control mice. Therefore the AdipoqCreER^T2 mouse line will be a valuable tool for studying the consequences of a temporally controlled deletion of floxed genes in white adipose tissue. genesis 48:618–625, 2010. © 2010 Wiley‐Liss, Inc.     

Cre-mediated recombination in cell lineages that express the progesterone receptor

Using gene-targeting methods, a progesterone receptor Cre knockin (PR-Cre) mouse was generated in which Cre recombinase was inserted into exon 1 of the PR gene. The insertion positions the Cre gene downstream (and under the specific control) of the endogenous PR promoter. As for heterozygotes for the progesterone receptor knockout (PRKO) mutation, mice heterozygous for the Cre knockin insertion are phenotypically indistinguishable from wildtype. Crossing the PR-Cre with the ROSA26R reporter revealed that Cre excision activity is restricted to cells that express PR in progesterone-responsive tissues such as the uterus, ovary, oviduct, pituitary gland, and mammary gland. Initial characterization of the PR-Cre mouse underscores the utility of this model to precisely ablate floxed target genes specifically in cell lineages that express the PR. In the wider context of female reproductive tissue ontology, this model will be indispensable in tracing the developmental fate of cell lineages that descend from PR positive progenitors.     

Generation of a reporter mouse line expressing Akt and EGFP upon Cre-mediated recombination

The serine-threonine kinase Akt regulates multiple biological processes. An important strategy to study Akt signaling in different tissues is targeted activation of this pathway in vivo. The current studies describe the generation of a mouse model that combines a double reporter system with activation of a constitutively active form of Akt1 (caAkt) in a Cre-dependent manner. Before Cre recombination, these mice express LacZ during development as well as in most adult tissues. After Cre-mediated excision of the LacZ reporter, functionality of the transgene was demonstrated by expression of the caAkt mutant along with the second reporter, EGFP in different pancreatic compartments and in the nervous system. This animal model provides a critical reagent for assessing the effects of Akt activation in specific tissues. The lineage-tracing properties provide a useful tool to study the role of Akt signaling in regulation of differentiation programs during development and plasticity of mature tissues.     

肠道上皮与肠道微生物相互作用的研究现状

肠道上皮是肠上皮细胞及其分泌物有机构成的黏膜界面。随着技术的进步和对肠道菌群作用的逐渐重视,研究者对肠道上皮与肠道微生物相互作用的认识也不断深入。研究表明,肠道上皮调节并维持肠道微生物的定殖与分布,肠道微生物也影响肠道上皮的多种屏障功能,二者通过一系列细胞分子机制紧密联系,共同维持肠道稳态。此外,其过程中产生的宿主-肠道菌群共代谢物被发现可以反映宿主的生理病理状态,作为指标被应用于临床疾病诊断、治疗效果评估和预后推测。本文基于近年的研究,综述了肠道上皮与肠道微生物的相互作用及其细胞分子机制,为进一步研究和临床应用总结了理论基础,并探讨了未来可能的研究方向。     

Gasdermin D (Gsdmd) is dispensable for mouse intestinal epithelium development

Members of the novel gene family Gasdermin (Gsdm) are exclusively expressed in a highly tissue-specific manner in the epithelium of skin and the gastrointestinal tract. Based on their expression patterns and the phenotype of the Gsdma3 spontaneous mutations, it is inferred that the Gsdm family genes are involved in epithelial cell growth and/or differentiations in different tissues. To investigate possible roles of the Gsdm gene family in the development of intestinal tracts, we generated a Gsdmd mutant mouse, which is a solitary member of the Gsdmd subfamily and which is predominantly expressed in the intestinal tract by means of targeted disruption. In the mutant homozygotes, we found no abnormality of intestinal tract morphology. Moreover, in mutant mice, there was normal differentiation of all constituent cell types of the intestinal epithelium. Thus, this study clearly shows that Gsdmd is not essential for development of mouse intestinal tract or epithelial cell differentiation.     

Murine intestinal organoids resemble intestinal epithelium in their microRNA profiles

Intestinal organoids were established as an ex vivo model of the intestinal epithelium. We investigated whether organoids resemble the intestinal epithelium in their microRNA (miRNA) profiles. Total RNA samples were obtained from crypt and villus fractions in murine intestine and from cultured organoids. Microarray analysis showed that organoids largely resembled intestinal epithelial cells in their miRNA profiles. In silico prediction followed by qRT-PCR suggested that six genes are regulated by corresponding miRNAs along the crypt-villus axis, suggesting miRNA regulation of epithelial cell renewal in the intestine. However, such expression patterns of miRNAs and their target mRNAs were not reproduced during organoids maturation. This might be due to lack of luminal factors and endocrine, nervous, and immune systems in organoids and different cell populations between in vivo epithelium and organoids. Nevertheless, we propose that intestinal organoids provide a useful in vitro model to investigate miRNA expression in intestinal epithelial cells.     

F9 embryonal carcinoma cells engineered for tamoxifen-dependent Cre-mediated site-directed mutagenesis and doxycycline-inducible gene expression

The study of gene functions in complex genetic environments such as mammalian cells would greatly benefit from systems allowing a tight control of gene expression. The tetracycline-inducible gene expression system and the site-specific Cre/loxP recombination system have gained increasing popularity for conditional expression and gene disruption. To facilitate the analysis of gene functions in a cell autonomous system, we have established an F9 murine embryonal carcinoma cell line, constitutively expressing both the doxycycline-controlled transactivator rtTA and the tamoxifen-dependent Cre recombinase Cre-ER(T). The expression of a reporter gene placed under the control of tetracycline operators was induced about 1000-fold by doxycycline, and tamoxifen-induced excision of a loxP-flanked DNA segment occurred in all cells. This genetically engineered cell line, which allows, upon simple ligand addition, sophisticated genetic manipulations, such as sequential inactivation of loxP-flanked genes, and tightly controlled reexpression of their cDNAs, should be a valuable tool for studying mammalian gene functions.     

Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction

Here we describe a triple transgenic mouse system, which combines the tissue specificity of any Cre-transgenic line with the inducibility of the reverse tetracycline transactivator (rtTA)/tetracycline-responsive element (tet-O)-driven transgenes. To ensure reliable rtTA expression in a broad range of cell types, we have targeted the rtTA transgene into the ROSA26 locus. The rtTA expression, however, is conditional to a Cre recombinase-mediated excision of a STOP region from the ROSA26 locus. We demonstrate the utility of this technology through the inducible expression of the vascular endothelial growth factor (VEGF-A) during embryonic development and postnatally in adult mice. Our results of adult induction recapitulate several different hepatic and immune cell pathological phenotypes associated with increased systemic VEGF-A protein levels. This system will be useful for studying genes in which temporal control of expression is necessary for the discovery of the full spectrum of functions. The presented approach abrogates the need to generate tissue-specific rtTA transgenes for tissues where well-characterized Cre lines already exist.     

Efficient temporally-controlled targeted mutagenesis in smooth muscle cells of the adult mouse

To generate temporally-controlled targeted somatic mutations selectively and efficiently in smooth muscles, we have established a transgenic SMA-Cre-ER(T2) mouse line in which the expression of the Tamoxifen-dependent Cre-ER(T2) recombinase is under the control of a large genomic DNA segment of the mouse smooth muscle alpha actin (SMA) gene, contained in a Bacterial artificial chromosome (Bac). In this transgenic mouse line, Cre-ER(T2)-mediated recombination of LoxP-flanked target DNA is strictly Tamoxifen-dependent, and efficient in both vascular and visceral smooth muscle cells. Moreover, with the exception of few cardiomyocytes, LoxP-flanked DNA excision is restricted to smooth muscle cells. Thus, SMA-Cre-ER(T2) mice should be of great value to analyze gene function in smooth muscles, and to establish new animal models of human smooth muscle disorders.     

Differentiation of proventricular epithelium in xenoplastic associations with mesenchymal or fibroblastic cells

Summary Proventricular epithelium (PV epithelium) from 6-day chicken embryos was associated with cultured cells, derived from fetal rat small intestine, or with fetal rat or human skin fibroblasts. The cytodifferentiation of PV epithelium was investigated using antibodies to chicken pepsinogen, a marker protein of PV epithelium, and to chicken sucrase, a marker enzyme of the small-intestinal brush-border membrane. PV epithelium formed complex glands and produced pepsinogen in association with cultured gut mesenchymal cells and skin fibroblasts. Its development was comparable to that achieved under the influence of PV mesenchyme. PV epithelial development was severely inhibited, however, under the influence of intact chicken or rat intestinal mesenchyme. The data are consistent with the idea that during the first step of epithelial-mesenchymal interactions, the epithelium and not the mesenchyme may be responsible for the determination of the developmental fate.     

Process development for an inducible rituximab-expressing Chinese hamster ovary cell line

Inducible mammalian expression systems are becoming increasingly available and are not only useful for the production of cytotoxic/cytostatic products, but also confer the unique ability to uncouple the growth and production phases. In this work, we have specifically investigated how the cell culture state at the time of induction influences the cumate-inducible expression of recombinant rituximab by a GS-CHO cell line. To this end, cells grown in batch and fed-batch cultures were induced at increasing cell densities (1 to 10 × 10 ⁶ cells/mL). In batch, the cell specific productivity and the product yield were found to reduce with increasing cell density at induction. A dynamic feeding strategy using a concentrated nutrient solution applied prior and postinduction allowed to significantly increase the integral of viable cells and led to a 3-fold increase in the volumetric productivity (1.2 g/L). The highest product yields were achieved for intermediate cell densities at induction, as cultures induced during the late exponential phase (10 × 10 ⁶ cells/mL) were associated with a shortened production phase. The final glycosylation patterns remained however similar, irrespective of the cell density at induction. The kinetics of growth and production in a 2 L bioreactor were largely comparable to shake flasks for a similar cell density at induction. The degree of galactosylation was found to decrease over time, but the final glycan distribution at harvest was consistent to that of the shake flasks cultures. Taken together, our results provide useful insights for the rational development of fed-batch cell culture processes involving inducible CHO cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2742, 2019     

Ciliated epithelium in the gut of larval Atlantic halibut, Hippoglossus hippoglossus

The presence of cilia was documented in the hindgut of larval Atlantic halibut ( Hippoglossus hippoglossus ) with the use of electron microscopy. Cilia were shown to be present as late as 8 and 9 day post hatch. These observations were compared to similar observations in more primitive teleosts.     

A mart-1::Cre transgenic line induces recombination in melanocytes and retinal pigment epithelium

The number of transgenic mouse lines expressing Cre in either type of pigment cells (melanocytes and retinal pigment epithelium, RPE) is limited, and the available lines do not always offer sufficient specificity. In this study, we addressed this issue and we report on the generation of a MART‐1::Cre BAC transgenic mouse line, in which the expression of Cre recombinase is controlled by regulatory elements of the pigment cell‐specific gene MART‐1 (mlana). When MART‐1::Cre BAC transgenic mice were bred with the ROSA26‐R reporter line, ß‐galactosidase expression was observed in RPE from E12.5 onwards, and in melanocyte precursors from E17.5, indicating that the MART‐1::Cre line provides Cre recombinase activity in pigment‐producing cells rather than in a particular lineage. In addition, breeding of this mouse line to mice carrying a conditional allele of RBP‐Jκ corroborated the reported phenotypes in both pigment cell lineages, inducing hair greying and microphthalmia. Our results thus suggest, that the MART‐1::Cre line may serve as a novel and useful tool for functional studies in melanocytes and the RPE.genesis 49:403–409, 2011. © 2011 Wiley‐Liss, Inc.     

Cre-mediated recombination at the murine whey acidic protein (mWAP) locus

The mouse whey acidic protein (WAP) gene in mouse embryonic stem (ES) cells has been targeted with a loxP-flanked neomycin phosphotransferase-thymidine kinase (neo-TK) cassette inserted into exon 4. Southern blot revealed that 51 of 199 colonies were correctly targeted (1:4). Next, a Cre-encoding plasmid was electroporated into a targeted cell line to cause the deletion of the neo-TK cassette. Modified ES cell colonies were identified by polymerase chain reaction (PCR); 44 out of 50 colonies (88%) had undergone Cre-mediated deletion. Finally, a loxP-tagged cell line was co-electroporated with a Cre-encoding plasmid and a loxP-containing neo plasmid for site-specific insertion into the WAP locus. The frequency of this event was 23% (11 of 48) of that obtained with random integration. This demonstrates the feasibility of using the Cre-loxP system for site-specific integration in ES cells. Moreover, this is the first report of targeting a loxP-containing transgene into a predetermined location in ES cells. Ultimately, a mouse model derived from these modified ES cells will usher in a second generation of animal “bioreactor” models where the inserted transgene is controlled exclusively by the endogenous locus regulatory elements. In addition, oncogenesis can be explored from single copy oncogene/tumor suppressor gene inserts, which are regulated in a temporal and tissue-specific manner. It is hoped that regulation of transgene expression in this fashion will help elucidate the underlying mechanisms of normal development in the mammary gland. Mol. Reprod. Dev. 48:324–331, 1997. © 1997 Wiley-Liss, Inc.