Generation and characterization of alpha-chymase-Cre transgenic mice

The Cre-loxP technology allows the introduction of somatic gene alterations in a tissue and/or cell type specific manner. The development of transgenes that target Cre expression to specific cell types is a critical component in this system. Here, we describe the generation and characterization of transgenic mouse lines expressing Cre recombinase under the control of the baboon alpha-chymase promoter, designated Chm:Cre, in order to direct Cre expression specifically to mouse mast cells. Chm:Cre expression was detected in mast cells in lung and colon tissue. Cre-mediated recombination in these mice identified a population of mature tissue resident mast cells using ROSA26R reporter mice. No Cre-expression and Cre-mediated recombination was induced in in vitro generated bone marrow derived mast cells or mast cells isolated from the peritoneal cavity indicating that Cre-expression under the control of the alpha-chymase promoter is solely activated in tissue resident mast cells. These Chm:Cre transgenic mice represent a useful tool to specifically inactivate genes of interest in mast cells of these tissues.     

Generation and characterization of a GFP transgenic rat line for embryological research

Model organisms expressing fluorescent proteins are important tools for research. The present study was performed to generate and characterize a new line of green fluorescent protein (GFP) transgenic rats for use as a model in experimental embryological research. We injected a GFP expression vector into 135 zygotes of the Sprague-Dawley (SD) rat strain. Embryo transfer of 103 surviving embryos resulted in the production of 35 offspring (33.9%) and two of them were transgenic (5.7%). Two transgenic rat lines that ubiquitously express GFP under the control of the cytomegalovirus-enhancer/beta-actin (CAGGS) promoter were generated by breeding. We studied the main embryological parameters of one these GFP transgenic lines. Homozygous GFP-transgenic females have the same ovulation and superovulation rates as wild type (WT) females. Transgenic embryos reached blastocyst stage in vitro and developed in vivo after embryo transfer without decrease in their developmental ability compared to the control group. The genotype of the parents determined the onset of GFP expression in preimplantation embryos. When the GFP gene is derived from the transgenic female parent, fluorescence was detected in oocytes and in embryos of all further stages of development. When the GFP gene is inherited by the transgenic male parent, GFP was only expressed from the blastocyst stage on. GFP-transgenic rats represent a valuable tool to mark embryos for many embryological studies such as transgenesis, gene expression patterns during early development, embryo aggregation for analysis of the distribution of cells in chimeric embryos and nuclear transfer to confirm the origin of the cloned offspring.     

Generation and characterization of a conditionally immortalized lung clara cell line from the H-2Kb-tsA58 transgenic mouse

The Clara cell is believed to be the progenitor of the peripheral airway epithelium, and it produces the surfactant proteins SP-A and SP-B, in addition to the 10-kDa Clara cell secretory protein (CCSP or CC10). To date, attempts to develop Clara cell lines have been unsuccessful. Most such attempts have involved the in vitro insertion of a transforming viral oncogene. We have reported previously the characterization of a differentiated conditionally immortalized murine lung Type II epithelial cell line, T7, from the H-2Kb-tsA58 transgenic mouse. We have also used this mouse model to derive Clara cell lines. In this model, the need for in vitro gene insertion is circumvented by the creation of a transgene, in which the large tumor antigen of a temperature-sensitive strain (tsA58) of the simian virus 40 (SV40) is fused with the major histocompatibility complex promoter H-2Kb. The promoter is active in a wide range of tissues and is induced by interferons (IFN). From the lungs of animals harboring the hybrid construct, we isolated and characterized Clara cells. The cells contain dense secretory granules and mitochondria typical of Clara cells, and express SP-A, SP-B, SP-D, and the Clara cell secretory protein, CC10. Withdrawal of the IFN and elevation of the incubation temperature permit normal cell differentiation similar to that of Clara cells in vivo. This cell line should be very useful for the investigation of normal Clara cell function and gene expression.     

Generation and characterization of iUBC-KikGR photoconvertible transgenic mice for live time-lapse imaging during development

A transgenic mouse line named iUBC-KikGR was generated, which expresses the photoconvertible fluorescent protein Kikume Green-Red (KikGR) under the control of the human Ubiquitin C promoter. KikGR is natively a green fluorophore, which can be converted into a red fluorophore upon exposure to UV light. KikGR is expressed broadly throughout transgenic embryos from the two-cell stage onward and in the adult. Specificity of photoconversion can range from the entire embryo to a region of an organ, to a few individual cells, depending on the needs of the experimenter. Cell movements, tissue reorganization, and migration can then be observed in real time by culturing the tissue of interest as an explant on the microscope stage. The iUBC-KikGR transgenic line represents a singular genetic reagent, which can be used for fate mapping, lineage tracing, and live visualization of cell behaviors and tissue movements in multiple organs at multiple time points.     

Generation and characterization of Csrp1 enhancer-driven tissue-restricted Cre-recombinase mice

Cell type-specific genetic modification using the LoxP/Cre system is a powerful tool for genetic analysis of distinct cell lineages. Because of the unique arterial smooth muscle-restricted expression of a 5.0 kb cysteine-rich protein (Csrp1) enhancer (Lilly et al.,2001, Dev Biol 240:531-547), we hypothesized that a transgenic Cre line would prove useful for the smooth muscle lineage-specific genetic manipulation. Here we describe a transgenic mouse line, ECsrp1(Cre), where Cre is initially specifically expressed in arterial smooth muscle cells. Use of the ROSA26R reporter allele confirmed that Cre-mediated recombination in vascular smooth muscle cells began at approximately E10.0 and was highly proficient. Subsequently, Cre is expressed in restricted skeletal and nonvascular smooth muscle lineages. This lineage tracing data is important for future conditional knockout studies to understand where and when Cre-mediated deletion occurs and where Cre-expressing daughter cells finally localize. Additionally, we crossed the ECsrp1(Cre) mice to the ROSA26(-eGFP-DTA) diphtheria toxin A-expressing mice to genetically ablate ECsrp1(Cre) expressing cells. This ECsrp1(Cre) transgenic line should thus prove useful for genetic analysis of diverse aspects of cardiovascular morphogenesis and as a general smooth muscle lineage deletor line.     

A transgenic mouse that reveals cell shape and arrangement during ureteric bud branching

Understanding the cellular events that underlie epithelial morphogenesis is a key problem in developmental biology. Here, we describe a new transgenic mouse line that makes it possible to visualize individual cells specifically in the Wolffian duct and ureteric bud, the epithelial structures that give rise to the collecting system of the kidney. myr‐Venus, a membrane‐associated form of the fluorescent protein Venus, was expressed in the ureteric bud lineage under the control of the Hoxb7 promoter. In Hoxb7/myr‐Venus mice, the outlines of all Wolffian duct and ureteric bud epithelial cells are strongly labeled at all stages of urogenital development, allowing the shapes and arrangements of individual cells to be readily observed by confocal microscopy of freshly excised or cultured kidneys. This strain should be extremely useful for studies of cell behavior during ureteric bud branching morphogenesis in wild type and mutant mouse lines. genesis 47:61–66, 2009. © 2008 Wiley‐Liss, Inc.     

Generation of transgenic mice that conditionally express microRNA miR‐145

MicroRNAs are modulators of cellular phenotypes and their functions contribute to development, homeostasis, and disease. miR‐145 is a conserved microRNA that has been implicated in regulating an array of phenotypes. These include supporting smooth muscle differentiation, repression of stem cell pluripotency, and inhibition of tumor growth and metastasis. Previously, our lab demonstrated that miR‐145 acts to suppress cardiac fibrosis through inhibition of the TGF‐β signaling pathway. The range of effects that miR‐145 has on different cell types makes it an attractive microRNA for further study. Here we describe the generation of transgenic mice that conditionally express miR‐145 through Cre recombinase‐mediated activation. Characterization of individual founder lines indicates that overexpression of miR‐145 in the developing cardiovascular system has detrimental effects, with three independent miR‐145 transgenic lines exhibiting Cre‐dependent lethality. Expression analysis demonstrates that the transgene is robustly expressed and our analysis reveals a novel downstream target of miR‐145, Tnnt2. The miR‐145 transgenic mice represent a valuable tool to understand the role of miR‐145 in diverse cell types and to address its potential as a therapeutic mediator for the treatment of disease.     

A tetracycline‐inducible and skeletal muscle‐specific Cre recombinase transgenic mouse

We have generated a transgenic mouse that expresses Cre recombinase only in skeletal muscle and only following tetracycline treatment. This spatiotemporal specificity is achieved using two transgenes. The first transgene uses the human skeletal actin (HSA) promoter to drive expression of the reverse tetracycline‐controlled transactivator (rtTA). The second transgene uses a tetracycline responsive promoter to drive the expression of Cre recombinase. We monitored transgene expression in these mice by crossing them with ROSA26 loxP‐LacZ reporter mice, which express β‐galactosidase when activated by Cre. We find that the expression of this transgene is only detectable within skeletal muscle and that Cre expression in the absence of tetracycline is negligible. Cre is readily induced in this model with tetracycline analogs at a range of embryonic and postnatal ages and in a pattern consistent with other HSA transgenic mice. This mouse improves upon existing transgenic mice in which skeletal muscle Cre is expressed throughout development by allowing Cre expression to begin at later developmental stages. This temporal control of transgene expression has several applications, including overcoming embryonic or perinatal lethality due to transgene expression. This mouse is especially suited for studies of steroid hormone action, as it uses tetracycline, rather than tamoxifen, to activate Cre expression. In summary, we find that this transgenic induction system is suitable for studies of gene function in the context of hormonal regulation of skeletal muscle or interactions between muscle and motoneurons in mice. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Podocyte-specific expression of cre recombinase in transgenic mice

We report a transgenic mouse line that expresses Cre recombinase exclusively in podocytes. Twenty- four transgenic founders were generated in which Cre recombinase was placed under the regulation of a 2.5-kb fragment of the human NPHS2 promoter. Previously, this fragment was shown to drive beta-galactosidase (beta-gal) expression exclusively in podocytes of transgenic mice. For analysis, founder mice were bred with ROSA26 mice, a reporter line that expresses beta-gal in cells that undergo Cre recombination. Eight of 24 founder lines were found to express beta-gal exclusively in the kidney. Histological analysis of the kidneys showed that beta-gal expression was confined to podocytes. Cre recombination occurred during the capillary loop stage in glomerular development. No evidence for Cre recombination was detected in any of 14 other tissues examined.     

Generation of easily accessible human kidney tubules on two‐dimensional surfaces in vitro

The generation of tissue‐like structures in vitro is of major interest for various fields of research including in vitro toxicology, regenerative therapies and tissue engineering. Usually 3D matrices are used to engineer tissue‐like structures in vitro, and for the generation of kidney tubules, 3D gels are employed. Kidney tubules embedded within 3D gels are difficult to access for manipulations and imaging. Here we show how large and functional human kidney tubules can be generated in vitro on 2D surfaces, without the use of 3D matrices. The mechanism used by human primary renal proximal tubule cells for tubulogenesis on 2D surfaces appears to be distinct from the mechanism employed in 3D gels, and tubulogenesis on 2D surfaces involves interactions between epithelial and mesenchymal cells. The process is induced by transforming growth factor‐β₁, and enhanced by a 3D substrate architecture. However, after triggering the process, the formation of renal tubules occurs with remarkable independence from the substrate architecture. Human proximal tubules generated on 2D surfaces typically have a length of several millimetres, and are easily accessible for manipulations and imaging, which makes them attractive for basic research and in vitro nephrotoxicology. The experimental system described also allows for in vitro studies on how primary human kidney cells regenerate renal structures after organ disruption. The finding that human kidney cells organize tissue‐like structures independently from the substrate architecture has important consequences for kidney tissue engineering, and it will be important, for instance, to inhibit the process of tubulogenesis on 2D surfaces in bioartificial kidneys.     

Conditional tamoxifen Cre induced mutagenesis in the embryonic kidney in organ culture

Developmental control genes are often sequentially and repeatedly functional during embryogenesis, and for this reason conditional mutagenesis tools are often required to study their roles in detail. Cre recombinase fused to the modified estrogen hormone-binding domain (ER(Tm)) generates a Cre in which the recombination activity of the LoxP-containing gene can be regulated by the nonsteroidal estrogen analogue 4-hydroxytamoxifen (4OH-TM). ER(Tm) may provide a useful way of achieving conditional mutagenesis in conjunction with the classic organ culture methods of experimental embryology. We used embryonic kidneys separated from the Cre-ER(Tm); R26R embryos to assay whether efficient 4OH-TM-inducible genomic recombination can be achieved in organ culture and in experimentally induced kidney mesenchymes. Our results indicate that the inducible ER(Tm) Cre/loxP system indeed provides an effective way of conditionally mutagenizing genes in kidney organ culture and tissue conjugates.     

Plasticity of distal nephron epithelia from human kidney organoids enables the induction of ureteric tip and stalk

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Capn8 promoter directs the expression of Cre recombinase in gastric pit cells of transgenic mice

Gastric pit cells are high‐turnover epithelial cells of the gastric mucosa. They secrete mucus to protect the gastric epithelium from acid and pepsin. To investigate the genetic mechanisms underlying the physiological functions of gastric pit cells, we generated a transgenic mouse line, namely, Capn8‐Cre, in which the expression of Cre recombinase was controlled by the promoter of the intracellular Ca²⁺‐regulated cysteine protease calpain‐8. To test the tissue distribution and excision activity of Cre recombinase, the Capn8‐Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4^Co/Co). Multiple‐tissue PCR and LacZ staining demonstrated that Capn8‐Cre transgenic mouse expressed Cre recombinase in the gastric pit cells. Cre recombinase activity was also detected in the liver and skin tissues. These data suggest that the Capn8‐Cre mouse line described here could be used to dissect gene function in gastric pit cells. genesis 47:674–679, 2009. © 2009 Wiley‐Liss, Inc.