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.     

Generation of Smad7‐Cre recombinase mice: A useful tool for the study of epithelial‐mesenchymal transformation within the embryonic heart

Smad7 can be induced by various transforming growth factor‐β superfamily ligands and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. Previous analyses have demonstrated that although Smad7 is widely expressed, it is predominantly found in the vascular endothelium. Because of the restricted spatiotemporal reporter expression driven via a novel 4.3 kb Smad7 promoter in endocardial cells overlying the hearts atrioventricular (AV) cushions; we hypothesized that a transgenic Cre line would prove useful for the analysis of endocardial cushion and valve formation. Here we describe a mouse line, Smad7^Cre, where Cre is robustly expressed within both cardiac outflow and AV endocardial cushions. Additionally, as endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme, we crossed the Smad7^Cre mice to the ROSA26^eGFP‐DTA diphtheria toxin A‐expressing mice in order to genetically ablate Smad7^Cre expressing cells. Ablation of Smad7^Cre cells resulted in embryonic lethality by E11.5 and largely acellular endocardial cushions. genesis 47:469–475, 2009. © 2009 Wiley‐Liss, Inc.     

A new tool for conditional gene manipulation in a subset of keratin‐expressing epithelia

Megsin is a serine protease inhibitor (Serpin) that has known expression in kidney mesangial cells. Here, we report the generation and characterization of a bacterial artificial chromosome (BAC) transgene expressing Cre under the control of Megsin regulatory elements. When crossed to the ROSA26R‐lacZ reporter mice, the Megsin‐Cre transgene mediates loxP recombination primarily in the skin, forestomach, and esophagus, but surprisingly not in the mesangial cells. Within the skin, cells in all epidermal layers and the hair follicle cells expressed Cre. This transgene also has uniform expression in the epithelium of the forestomach and esophagus. Conditional deletion of Adam10, a gene known to have important functions in skin development, by using this Megsin‐Cre transgene led to severe skin defects. In addition, these mutants appear to have reduced folds and surface area in the forestomach. These results show that the Megsin‐Cre transgene can mediate loxP‐recombination in all epidermal layers of the skin, the hair follicle cells, as well as in the epithelium of the forestomach and esophagus, all of which have known expression of various keratins. This Megsin‐Cre transgene can serve as a new tool for conditional genetic manipulation to study development and diseases in the skin and the upper digestive tract. genesis 50:899–907, 2012. © 2012 Wiley Periodicals, Inc.     

Inducible cardiomyocyte‐specific gene disruption directed by the rat Tnnt2 promoter in the mouse

We developed a conditional and inducible gene knockout methodology that allows effective gene deletion in mouse cardiomyocytes. This transgenic mouse line was generated by coinjection of two transgenes, a “reverse” tetracycline‐controlled transactivator (rtTA) directed by a rat cardiac troponin T (Tnnt2) promoter and a Cre recombinase driven by a tetracycline‐responsive promoter (TetO). Here, Tnnt2‐rtTA activated TetO‐Cre expression takes place in cardiomyocytes following doxycycline treatment. Using two different mouse Cre reporter lines, we demonstrated that expression of Cre recombinase was specifically and robustly induced in the cardiomyocytes of embryonic or adult hearts following doxycycline induction, thus, allowing cardiomyocyte‐specific gene disruption and lineage tracing. We also showed that rtTA expression and doxycycline treatment did not compromise cardiac function. These features make the Tnnt2‐rtTA;TetO‐Cre transgenic line a valuable genetic tool for analysis of spatiotemporal gene function and cardiomyocyte lineage tracing during developmental and postnatal periods. genesis 48:63–72, 2010. © 2009 Wiley‐Liss, Inc.     

A genetic link between Tbx1 and fibroblast growth factor signaling

Tbx1 haploinsufficiency causes aortic arch abnormalities in mice because of early growth and remodeling defects of the fourth pharyngeal arch arteries. The function of Tbx1 in the development of these arteries is probably cell non-autonomous, as the gene is not expressed in structural components of the artery but in the surrounding pharyngeal endoderm. We hypothesized that Tbx1 may trigger signals from the pharyngeal endoderm directed to the underlying mesenchyme. We show that the expression patterns of Fgf8 and Fgf10, which partially overlap with Tbx1 expression pattern, are altered in Tbx1(-/-) mutants. In particular, Fgf8 expression is abolished in the pharyngeal endoderm. To understand the significance of this finding for the pathogenesis of the mutant Tbx1 phenotype, we crossed Tbx1 and Fgf8 mutants. Double heterozygous Tbx1(+/-);Fgf8(+/-) mutants present with a significantly higher penetrance of aortic arch artery defects than do Tbx1(+/-);Fgf8(+/+) mutants, while Tbx1(+/+);Fgf8(+/-) animals are normal. We found that Fgf8 mutation increases the severity of the primary defect caused by Tbx1 haploinsufficiency, i.e. early hypoplasia of the fourth pharyngeal arch arteries, consistent with the time and location of the shared expression domain of the two genes. Hence, Tbx1 and Fgf8 interact genetically in the development of the aortic arch. Our data provide the first evidence of a genetic link between Tbx1 and FGF signaling, and the first example of a modifier of the Tbx1 haploinsufficiency phenotype. We speculate that the FGF8 locus might affect the penetrance of cardiovascular defects in individuals with chromosome 22q11 deletions involving TBX1.     

Dwarfism in homozygous Agc1CreERT mice is associated with decreased expression of aggrecan

Aggrecan (Acan), a large proteoglycan is abundantly expressed in cartilage tissue. Disruption of Acan gene causes dwarfism and perinatal lethality of homozygous mice. Because of sustained expression of Acan in the growth plate and articular cartilage, Agc^Cre model has been developed for the regulated ablation of target gene in chondrocytes. In this model, the IRES‐CreERT‐Neo‐pgk transgene is knocked‐in the 3′UTR of the Acan gene. We consistently noticed variable weight and size among the Agc^Cre littermates, prompting us to examine the cause of this phenotype. Wild‐type, Cre‐heterozygous (Agc^+/Cre), and Cre‐homozygous (Agc^Cre/Cre) littermates were indistinguishable at birth. However, by 1‐month, Agc^Cre/Cre mice showed a significant reduction in body weight (18–27%) and body length (19–22%). Low body weight and dwarfism was sustained through adulthood and occurred in both genders. Compared with wild‐type and Agc^+/Cre littermates, long bones and vertebrae were shorter in Agc^Cre/Cre mice. Histological analysis of Agc^Cre/Cre mice revealed a significant reduction in the length of the growth plate and the thickness of articular cartilage. The amount of proteoglycan deposited in the cartilage of Agc^Cre/Cre mice was nearly half of the WT littermates. Analysis of gene expression indicates impaired differentiation of chondrocyte in hyaline cartilage of Agc^Cre/Cre mice. Notably, both Acan mRNA and protein was reduced by 50% in Agc^Cre/Cre mice. A strong correlation was noted between the level of Acan mRNA and the body length. Importantly, Agc^+/Cre mice showed no overt skeletal phenotype. Thus to avoid misinterpretation of data, only the Agc^+/Cre mice should be used for conditional deletion of a target gene in the cartilage tissue.     

Generation and characterization of a conditional allele of Interferon Regulatory Factor 6

Interferon Regulatory Factor 6 (IRF6) is a critical regulator of differentiation, proliferation, and migration of keratinocytes. Mutations in IRF6 cause two autosomal dominant disorders characterized by cleft lip with or without cleft palate. In addition, DNA variation in IRF6 confers significant risk for non‐syndromic cleft lip and palate. IRF6 is also implicated in adult onset development and disease processes, including mammary gland development and squamous cell carcinoma. Mice homozygous for a null allele of Irf6 die shortly after birth due to severe skin, limb, and craniofacial defects, thus impeding the study of gene function after birth. To circumvent this, a conditional allele of Irf6 was generated. To validate the functionality of the conditional allele, we used three “deleter” Cre strains: Gdf9‐Cre, CAG‐Cre, and Ella‐Cre. When Cre expression was driven by the Gdf9‐Cre or CAG‐Cre transgenes, 100% recombination was observed as indicated by DNA genotyping and phenotyping. In contrast, use of the Ella‐Cre transgenic line resulted in incomplete recombination, despite expression at the one‐cell stage. In sum, we generated a novel tool to delete Irf6 in a tissue specific fashion, allowing for study of gene function past perinatal stages. However, recombination efficiency of this allele was dictated by the Cre‐driver used.     

Cre/ lox site-specific recombination controls the excision of a transgene from the rice genome

The Cre/lox site-specific recombination controls the excision of a target DNA segment by recombination between two lox sites flanking it, mediated by the Cre recombinase. We have studied the functional expression of the Cre/lox system to excise a transgene from the rice genome. We developed transgenic plants carrying the target gene, hygromycin phosphotransferase (hpt) flanked by two lox sites and transgenic plants harboring the Cre gene. Each lox plant was crossed with each Cre plant reciprocally. In the Cre/lox hybrid plants, the Cre recombinase mediates recombination between two lox sites, resulting in excision of the hpt gene. The recombination event could be detected because it places the CaMV 35S promoter of the hpt gene adjacent to a promoterless gusA gene; as a result the gusA gene is activated and its expression could be visualized. In 73 Cre/lox hybrid plants from various crosses of T0 transgenic plants, 19 expressed GUS, and in 132 Cre/lox hybrid plants from crosses of T2 transgenic plants, 77 showed GUS expression. Molecular data proved the excision event occurred in all the GUS⁺ plants. Recombination occurred with high efficiency at the early germinal stage, or randomly during somatic development stages. Received. 2 April 2001 / Accepted: 29 June 2001     

Generation of mice with a conditional allele for G6pc

Glucose‐6‐phosphatase‐α (G6Pase‐α or G6PC) catalyzes the hydrolysis of glucose‐6‐phosphate to glucose and is a key enzyme in interprandial glucose homeostasis. Mutations in the human G6PC gene, expressed primarily in the liver, kidney, and intestine, cause glycogen storage disease Type Ia (GSD‐Ia), an autosomal recessive disorder characterized by a disturbed glucose homeostasis. For better understanding of the roles of G6Pase‐α in different tissues and in pathological conditions, we have generated mice harboring a conditional null allele for G6pc by flanking Exon 3 of the G6pc gene with loxP sites. We confirmed the null phenotype by using the EIIa‐Cre transgenic approach to generate mice lacking Exon 3 of the G6pc gene. The resulting homozygous Cre‐recombined null mice manifest a phenotype mimicking G6Pase‐α‐deficient mice and human GSD‐Ia patients. This G6pc conditional null allele will be valuable to examine the consequence of tissue‐specific G6Pase‐α deficiency and the mechanisms of long‐term complications in GSD‐Ia. genesis 47:590–594, 2009. Published 2009 Wiley‐Liss, Inc.     

Misexpression of Gbx2 throughout the mesencephalon by a conditional gain‐of‐function transgene leads to deletion of the midbrain and cerebellum in mice

The mouse homeobox gene, Gbx2, is expressed in discreet domains in the neural tube and plays a key role in forebrain and hindbrain development. Previous studies have demonstrated that mutual inhibition between Gbx2 and Otx2, which are respectively expressed in the anterior and posterior parts of the neural plate, positions the prospective midbrain–hindbrain junction. We describe here a conditional Gbx2 gain‐of‐function transgenic mouse line, Gbx2‐GOF, which expresses Gbx2 and red fluorescence protein, mCherry, upon Cre‐mediated recombination. In the absence of Cre, β‐galactosidase is broadly expressed in mouse embryos and adult brains carrying the transgene. By combining Gbx2‐GOF and En1^Cre knock‐in allele, we activated expression of Gbx2 and mCherry throughout the mesencephalon (mes) and rhombomere 1 (r1). The ectopic expression of Gbx2 causes an anterior shift of the mes/r1 junction at embryonic day 10.5. Interestingly, we found that persistent expression of Gbx2 throughout the mes/r1 region largely abolishes expression of the isthmic organizer gene Fgf8, leading to deletion of the midbrain and cerebellum at later stages. Our data suggest that the juxtaposition of the expression domains of Gbx2 and Otx2 within the mes/r1 area is essential for the maintenance of Fgf8 expression. Furthermore, the Gbx2‐GOF transgenic line is suitable for functional study of Gbx2 during development. genesis 47:667–673, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Generation and validation of mice carrying a conditional allele of the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is important for normal homeostasis in a variety of tissues and, when abnormally expressed or mutated, contributes to the development of many diseases. However, in vivo functional studies are hindered by the lack of adult mice lacking EGFR because of the pre‐ and postnatal lethality of EGFR deficient mice. We generated a conditional allele of Egfr (Egfr^tm1Dwt) by flanking exon 3 with loxP sites in order to investigate tissue‐specific functions of this widely expressed receptor tyrosine kinase. The activity of the Egfr^tm1Dwt allele is indistinguishable from wildtype Egfr. Conversely, the Egfr^Δ allele, generated by Cre‐mediated deletion of exon 3 using the germline EIIa‐Cre transgenic line, functions as a null allele. Egfr^Δ/Δ embryos that have complete ablation of EGFR activity and die at mid‐gestation with placental defects identical to those reported for mice homozygous for the Egfr^tm1Mag null allele. We also inactivated the Egfr^tm1Dwt allele tissue‐specifically in the skin epithelium using the K14‐Cre transgenic line. These mice were viable but exhibited wavy coat hair remarkably similar to mice homozygous for the Egfr^wa2 hypomorphic allele or heterozygous for the Egfr^Wa5 antimorphic allele. These results suggest that the hairless phenotype of Egfr nullizygous mice is not solely due to absence of EGFR in the epithelium, but that EGFR activity is required also in skin stromal cells for normal hair morphogenesis. This new mouse model should have wide utility to inactivate Egfr conditionally for functional analysis of EGFR in adult tissues and disease states. genesis 47:85–92, 2009. © 2008 Wiley‐Liss, Inc.