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.     

Incomplete cre‐mediated excision leads to phenotypic differences between Stra8‐iCre; Mov10l1lox/lox and Stra8‐iCre; Mov10l1lox/Δ mice

In the Cre–loxp system, expression level and activity of Cre recombinase in a Cre deleter line are critical because these determine not only the cell specificity of gene knockout (KO), but also the efficiency of Cre‐mediated excision in a specific cell lineage. Although the spatiotemporal expression pattern of a Cre transgene is usually defined upon the generation of the mouse line, the Cre excision efficiency in a specific targeted cell lineage is rarely evaluated and often assumed to be 100%. Incomplete excision can lead to highly variable phenotypes owing to mosaicism (i.e., coexistence of cells with the flox or the recombined flox allele) and this problem has long been overlooked. Here, we report that Stra8‐codon‐improved Cre recombinase (iCre), a transgenic allele expressing iCre under the control of the male germ cell‐specific Stra8 promoter, could efficiently delete one Mov10l1 flox allele in spermatogenic cells, whereas the excision was incomplete when two Mov10l1 flox alleles were present. The incomplete Cre‐mediated excision led to a testicular phenotype that was much less severe than that in the true conditional KO (inactivation, 100%) mice. Our findings suggest that it is essential to determine the efficiency of Cre excision when Cre–loxp system is used for deleting genes in a specific cell lineage and the Cre; gene^lox/Δ genotype should be used to evaluate phenotypes instead of Cre; gene^lox/lox owing to the fact that the latter usually bears incomplete deletion of the flox allele(s). genesis 51:481–490. © 2013 Wiley Periodicals, Inc.     

Postnatal male germ‐cell expression of cre recombinase in Tex101‐iCre transgenic mice

We have generated a transgenic mouse line that expresses improved Cre recombinase (iCre) under the control of the testis‐expressed gene 101 (Tex101) promoter. This transgenic mouse line was named Tex101‐iCre. Using the floxed ROSA reporter mice, we found that robust Cre recombinase activity was detected in postnatal testes with weak or no activity in other tissues. Within the testis, Cre recombinase was active in spermatogenic cells as early as the prospermatogonia stage at day 1 after birth. In 30‐ and 60‐day‐old mice, positive Cre recombinase activity was detected not only in prospermatogonia but also in spermatogenic cells at later stages of spermatogenesis. There was little or no Cre activity in interstitial cells. Breeding wild‐type females with homozygous floxed fibroblast growth factor receptor 2 (Fgfr2) males carrying the Tex101‐iCre transgene did not produce any progeny with the floxed Fgfr2 allele. All the progeny inherited a recombined Fgfr2 allele, indicating that complete deletion of the floxed Fgfr2 allele by Tex101‐iCre can be achieved in the male germline. Furthermore, FGFR2 protein was not detected in spermatocytes and spermatids of adult Fgfr2^fl/fl;Tex101‐iCre mice. Taken together, our results suggest that the Tex101‐iCre mouse line allows the inactivation of a floxed gene in spermatogenic cells in adult mice, which will facilitate the functional characterization of genes in normal spermatogenesis and male fertility. genesis 48:717–722, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Generation of PECAM‐1 (CD31) conditional knockout mice

Platelet endothelial cell adhesion molecule 1 (PECAM‐1) is an adhesion and signaling receptor that is expressed on endothelial and hematopoietic cells and plays important roles in angiogenesis, vascular permeability, and regulation of cellular responsiveness. To better understanding the tissue specificity of PECAM‐1 functions, we generated mice in which PECAM1, the gene encoding PECAM‐1, could be conditionally knocked out. A targeting construct was created that contains loxP sites flanking PECAM1 exons 1 and 2 and a neomycin resistance gene flanked by flippase recognition target (FRT) sites that was positioned upstream of the 3′ loxP site. The targeting construct was electroporated into C57BL/6 embryonic stem (ES) cells, and correctly targeted ES cells were injected into C57BL/6 blastocysts, which were implanted into pseudo‐pregnant females. Resulting chimeric animals were bred with transgenic mice expressing Flippase 1 (FLP1) to remove the FRT‐flanked neomycin resistance gene and mice heterozygous for the floxed PECAM1 allele were bred with each other to obtain homozygous PECAM1 ^flox/flox offspring, which expressed PECAM‐1 at normal levels and had no overt phenotype. PECAM1 ^flox/flox mice were bred with mice expressing Cre recombinase under the control of the SRY‐box containing gene 2 (Sox2Cre) promoter to delete the floxed PECAM1 allele in offspring (Sox2Cre;PECAM1 ^del/WT), which were crossbred to generate Sox2Cre; PECAM1 ^del/del offspring. Sox2Cre; PECAM1 ^del/del mice recapitulated the phenotype of conventional global PECAM‐1 knockout mice. PECAM1 ^flox/flox mice will be useful for studying distinct roles of PECAM‐1 in tissue specific contexts and to gain insights into the roles that PECAM‐1 plays in blood and vascular cell function.     

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.     

Generation of Elf5‐Cre knockin mouse strain for trophoblast‐specific gene manipulation

Placental development is a complex and highly controlled process during which trophoblast stem cells differentiate to various trophoblast subtypes. The early embryonic death of systemic gene knockout models hampers the investigation of these genes that might play important roles during placentation. A trophoblast specific Cre mouse model would be of great help for dissecting out the potential roles of these genes during placental development. For this purpose, we generate a transgenic mouse with the Cre recombinase inserted into the endogenous locus of Elf5 gene that is expressed specifically in placental trophoblast cells. To analyze the specificity and efficiency of Cre recombinase activity in Elf5‐Cre mice, we mated Elf5‐Cre mice with Rosa26^mT/mG reporter mice, and found that Elf5‐Cre transgene is expressed specifically in the trophoectoderm as early as embryonic day 4.5 (E4.5). By E12.5, the activity of Elf5‐Cre transgene was detected exclusively in all derivatives of trophoblast lineages, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. In addition, Elf5‐Cre transgene was also active during spermatogenesis, from spermatids to mature sperms, which is consistent with the endogenous Elf5 expression in testis. Collectively, our results provide a unique tool to delete specific genes selectively and efficiently in trophoblast lineage during placentation.     

Generation of BAF53b‐Cre transgenic mice with pan‐neuronal Cre activities

Molecular and functional studies of genes in neurons in mouse models require neuron‐specific Cre lines. The current available neuronal Cre transgenic or knock‐in lines either result in expression in a subset of neurons or expression in both neuronal and non‐neuronal tissues. Previously we identified BAF53b as a neuron‐specific subunit of the chromatin remodeling BAF complexes. Using a bacteria artificial chromosome (BAC) construct containing the BAF53b gene, we generated a Cre transgenic mouse under the control of BAF53b regulatory elements. Like the endogenous BAF53b gene, we showed that BAF53b‐Cre is largely neuron‐specific. In both central and peripheral nervous systems, it was expressed in all developing neurons examined and was not observed in neural progenitors or glial cells. In addition, BAF53b‐Cre functioned in primary cultures in a pan‐neuron‐specific manner. Thus, BAF53b‐Cre mice will be a useful genetic tool to manipulate gene expression in developing neurons for molecular, biochemical, and functional studies. genesis, 53:440–448, 2015. © 2015 Wiley Periodicals, 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.     

A new gain‐of‐function mouse line to study the role of Wnt3a in development and disease

Wnt/β‐catenin signals are important regulators of embryonic and adult stem cell self‐renewal and differentiation and play causative roles in tumorigenesis. Purified recombinant Wnt3a protein, or Wnt3a‐conditioned culture medium, has been widely used to study canonical Wnt signaling in vitro or ex vivo. To study the role of Wnt3a in embryogenesis and cancer models, we developed a Cre recombinase activatable Rosa26^Wnt3a allele, in which a Wnt3a cDNA was inserted into the Rosa26 locus to allow for conditional, spatiotemporally defined expression of Wnt3a ligand for gain‐of‐function (GOF) studies in mice. To validate this reagent, we ectopically overexpressed Wnt3a in early embryonic progenitors using the T‐Cre transgene. This resulted in up‐regulated expression of a β‐catenin/Tcf‐Lef reporter and of the universal Wnt/β‐catenin pathway target genes, Axin2 and Sp5. Importantly, T‐Cre; Rosa26^Wnt3a mutants have expanded presomitic mesoderm (PSM) and compromised somitogenesis and closely resemble previously studied T‐Cre; Ctnnb1^ex3 (β‐catenin^GOF) mutants. These data indicate that the exogenously expressed Wnt3a stimulates the Wnt/β‐catenin signaling pathway, as expected. The Rosa26^Wnt3a mouse line should prove to be an invaluable tool to study the function of Wnt3a in vivo.     

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.     

A mouse model with tamoxifen‐inducible thyrocyte‐specific cre recombinase activity

We have created a mouse model expressing tamoxifen‐inducible Cre recombinase (CreER^T2) under the control of the thyroglobulin (Tg) gene promoter to be able to study the role of defined genetic modifications in the regulation of thyroid function. We chose the thyroglobulin promoter, as it is expressed specifically in the thyroid. In order to obtain reliable expression under the control of the Tg promoter, we used a P1 artificial chromosome (PAC) containing a large piece of the Tg promoter. A tamoxifen inducible CreER^T2 construct was selected to avoid the possible consequences of the gene deletion for the development of the thyroid gland, and to study the role of gene deletion in the adult thyroid. Transgenic lines (TgCreER^T2) carrying this construct were generated and analyzed by crossing the TgCreER^T2 mice with the ROSA26LacZ reporter strain. The activity and specificity of the Cre recombinase was tested by staining for β‐galactosidase activity and by immunohistochemistry using an anti‐Cre‐antibody. In the TgCreER^T2xROSA26LacZ reporter line, Cre‐mediated recombination occurred specifically in the thyrocytes only after tamoxifen administration, and no significant staining was observed in controls. The recombination efficiency was nearly complete, since almost all thyrocytes showed X‐gal staining. We could also induce the recombination in utero by giving tamoxifen to the pregnant female. In addition, mice expressing TgCreER^T2 had no obvious histological changes, hormonal alterations, or different response to growth stimuli as compared to controls. These results demonstrate that the TgCreER^T2 mouse line is a powerful tool to study temporally controlled deletion of floxed genes in the thyroid. genesis 52:333–340, 2014. © 2014 Wiley Periodicals, Inc.     

Contrasting patterns of X‐chromosome divergence underlie multiple sex‐ratio polymorphisms in stalk‐eyed flies

Sex‐linked segregation distorters cause offspring sex ratios to differ from equality. Theory predicts that such selfish alleles may either go to fixation and cause extinction, reach a stable polymorphism or initiate an evolutionary arms race with genetic modifiers. The extent to which a sex ratio distorter follows any of these trajectories in nature is poorly known. Here, we used X‐linked sequence and simple tandem repeat data for three sympatric species of stalk‐eyed flies (Teleopsis whitei and two cryptic species of T. dalmanni) to infer the evolution of distorting X chromosomes. By screening large numbers of field and recently laboratory‐bred flies, we found no evidence of males with strongly female‐biased sex ratio phenotypes (SR) in one species but high frequencies of SR males in the other two species. In the two species with SR males, we find contrasting patterns of X‐chromosome evolution. T. dalmanni‐1 shows chromosome‐wide differences between sex‐ratio (X^SR) and standard (X^ST) X chromosomes consistent with a relatively old sex‐ratio haplotype based on evidence including genetic divergence, an inversion polymorphism and reduced recombination among X^SR chromosomes relative to X^ST chromosomes. In contrast, we found no evidence of genetic divergence on the X between males with female‐biased and nonbiased sex ratios in T. whitei. Taken with previous studies that found evidence of genetic suppression of sex ratio distortion in this clade, our results illustrate that sex ratio modification in these flies is undergoing recurrent evolution with diverse genomic consequences.     

A new Adamts9 conditional mouse allele identifies its non‐redundant role in interdigital web regression

ADAMTS9 is the most conserved member of a large family of secreted metalloproteases having diverse functions. Adamts9 null mice die before gastrulation, precluding investigations of its roles later in embryogenesis, in adult mice or disease models. We therefore generated a floxed Adamts9 allele to bypass embryonic lethality. In this mutant, unidirectional loxP sites flank exons 5–8, which encode the catalytic domain, including the protease active site. Mice homozygous for the floxed allele were viable, lacked an overt phenotype, and were fertile. Conversely, mice homozygous for a germ‐line deletion produced from the floxed allele by Cre‐lox recombination did not survive past gastrulation. Hemizygosity of the deleted Adamts9 in combination with mutant Adamts20 led to cleft palate and severe white spotting as previously described. Previously, Adamts9 haploinsufficiency combined with either Adamts20 or Adamts5 nullizygosity suggested a cooperative role in interdigital web regression, but the outcome of deletion of Adamts9 alone remained unknown. Here, Adamts9 was conditionally deleted in limb mesoderm using Prx1‐Cre mice. Unlike other ADAMTS single knockouts, limb‐specific Adamts9 deletion resulted in soft‐tissue syndactyly (STS) with 100% penetrance and concurrent deletion of Adamts5 increased the severity of STS. Thus, Adamts9 has both non‐redundant and cooperative roles in ensuring interdigital web regression. This new allele will be useful for investigating other biological functions of ADAMTS9. genesis 52:702–712, 2014. © 2014 Wiley Periodicals, Inc.     

Generation of a KOR‐Cre knockin mouse strain to study cells involved in kappa opioid signaling

The kappa opioid receptor (KOR) has numerous important roles in the nervous system including the modulation of mood, reward, pain, and itch. In addition, KOR is expressed in many non‐neuronal tissues. However, the specific cell types that express KOR are poorly characterized. Here, we report the development of a KOR‐Cre knockin allele, which provides genetic access to cells that express KOR. In this mouse, Cre recombinase (Cre) replaces the initial coding sequence of the Opkr1 gene (encoding the kappa opioid receptor). We demonstrate that the KOR‐Cre allele mediates recombination by embryonic day 14.5 (E14.5). Within the brain, KOR‐Cre shows expression in numerous areas including the cerebral cortex, nucleus accumbens and striatum. In addition, this allele is expressed in epithelium and throughout many regions of the body including the heart, lung, and liver. Finally, we reveal that KOR‐Cre mediates recombination of a subset of bipolar and amacrine cells in the retina. Thus, the KOR‐Cre mouse line is a valuable new tool for conditional gene manipulation to enable the study of KOR. genesis 54:29–37, 2016. © 2015 Wiley Periodicals, Inc.     

Neuropilin‐2 genomic elements drive cre recombinase expression in primitive blood,vascular and neuronal lineages

We have established a novel Cre mouse line, using genomic elements encompassing the Nrp2 locus, present within a bacterial artificial chromosome clone. By crossing this Cre driver line to R26R LacZ reporter mice, we have documented the temporal expression and lineage traced tissues in which Cre is expressed. Nrp2‐Cre drives expression in primitive blood cells arising from the yolk sac, venous and lymphatic endothelial cells, peripheral sensory ganglia, and the lung bud. This mouse line will provide a new tool to researchers wishing to study the development of various tissues and organs in which this Cre driver is expressed, as well as allow tissue‐specific knockout of genes of interest to study protein function. This work also presents the first evidence for expression of Nrp2 protein in a mesodermal progenitor with restricted hematopoietic potential, which will significantly advance the study of primitive erythropoiesis. genesis 53:709–717, 2015. © 2015 Wiley Periodicals, Inc.