Differential oocyte-specific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2Cre transgenic mice

Oocyte-specific deletion of ovarian genes using Cre/loxP technology provides an excellent tool to understand their physiological roles during folliculogenesis, oogenesis, and preimplantation embryonic development. We have generated a transgenic mouse line expressing improved Cre recombinase (iCre) driven by the mouse growth differentiation factor-9 (GDF-9) promoter. The resulting transgenic mouse line was named GDF-9-iCre mice. Using the floxed ROSA reporter mice, we found that Cre recombinase was expressed in postnatal ovaries, but not in heart, liver, spleen, kidney, and brain. Within the ovary, the Cre recombinase was exclusively expressed in the oocytes of primordial follicles and follicles at later developmental stages. The expression of iCre of GDF-9-iCre mice was shown to be earlier than the Cre expression of Zp3Cre and Msx2Cre mice, in which the Cre gene is driven by zona pellucida protein 3 (Zp3) promoter and a homeobox gene Msx2 promoter, respectively, in the postnatal ovary. Breeding wild-type males with heterozygous floxed germ cell nuclear factor (GCNF) females carrying the GDF-9-iCre transgene did not produce any progeny having the floxed GCNF allele, indicating that complete deletion of the floxed GCNF allele can be achieved in the female germline by GDF-9-iCre mice. These results suggest that GDF-9-iCre mouse line provides an excellent genetic tool for understanding functions of oocyte-expressing genes involved in folliculogenesis, oogenesis, and early embryonic development. Comparison of the ontogeny of the Cre activities of GDF-9-iCre, Zp3Cre, and Msx2Cre transgenic mice shows there is sequential Cre activity of the three transgenes that will allow inactivation of a target gene at different points in folliculogenesis.     

Two distinct pathways for O-fucosylation of epidermal growth factor-like or thrombospondin type 1 repeats

Epidermal growth factor-like (EGF) repeats and thrombospondin type 1 repeats (TSRs) are both small cysteine-knot motifs known to be O-fucosylated. The enzyme responsible for the addition of O-fucose to EGF repeats, protein O-fucosyltransferase 1 (POFUT1), has been identified and shown to be essential in Notch signaling. Fringe, an O-fucose beta1,3-N-acetylglucosaminyltransferase, elongates O-fucose on specific EGF repeats from Notch to form a disaccharide that can be further elongated to a tetrasaccharide. TSRs are found in many extracellular matrix proteins and are involved in protein-protein interactions. The O-fucose moiety on TSRs can be further elongated with glucose to form a disaccharide. The discovery of O-fucose on TSRs raised the question of whether POFUT1, or a different enzyme, adds O-fucose to TSRs. Here we demonstrate the existence of a TSR-specific O-fucosyltransferase distinct from POFUT1. Similar to POFUT1, the novel TSR-specific O-fucosyltransferase is a soluble enzyme that requires a properly folded TSR as an acceptor substrate. In addition, we found that a previously identified fucose-specific beta1,3-glucosyltransferase adds glucose to O-fucose on TSRs, but it does not modify O-fucose on an EGF repeat. Similarly, Lunatic fringe, Manic fringe, and Radical fringe are all capable of modifying O-fucose on an EGF repeat, but not on a TSR. Taken together, these results suggest that two distinct O-fucosylation pathways exist in cells, one specific for EGF repeat and the other for TSRs.     

Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice

We have generated a transgenic mouse line,Tg(Stra8-cre)1Reb (Stra8-cre), which expresses improved Cre recombinase under the control of a 1.4 Kb promoter region of the germ cell-specific stimulated by retinoic acid gene 8 (Stra8). cre is expressed only in males beginning at postnatal day (P)3 in early-stage spermatogonia and is detected through preleptotene-stage spermatocytes. To further define when cre becomes active, we crossed Stra8-cre males with Tg(ACTB-Bgeo/GFP)21Lbe (Z/EG) reporter females and compared the expression of enhanced green fluorescent protein (EGFP) with the protein encoded by the zinc finger and BTB domain containing 16 (Zbtb16) gene, PLZF-a marker for undifferentiated spermatogonia. Co-expression of EGFP is observed in the majority of PLZF+ cells. We also tested recombination efficiency by mating Stra8-cre;Z/EG males and females with wild-type mice and examining EGFP expression in the offspring. Recombination is detected in >95% of Z/EG+ pups born to Stra8-cre;Z/EG fathers but in none of the offspring born to transgenic mothers, a verification that cre is not functional in females. The postnatal, premeiotic, male germ cell-specific activity of Stra8-cre makes this mouse line a unique resource to study testicular germ cell development.     

Cre-conditional expression of constitutively active Notch1 in transgenic mice

The Notch signaling pathway plays a critical role during mammalian development. To bypass embryonic lethality associated with constitutive Notch1 signaling, we created transgenic mice with a floxed beta-geo/stop signal between a cytomegalo virus promoter and the constitutively active intracellular domain of Notch1 (IC-Notch1). IC-Notch1 is activated upon introduction of Cre recombinase and it is coexpressed with an enhanced green fluorescent protein or human placental alkaline phosphatase reporter. We created three IC-Notch1 transgenic mouse lines and crossed them to a general Cre deletor mouse line, pCX-Cre. The double transgenic IC-Notch1/pCX-Cre embryos have widespread expression of IC-Notch1 and reporters and die before 10.5 days of gestation. Morphological and histological analysis of the double transgenic embryos indicated growth arrest and various developmental defects, including lack of neural tube closure, disorganized somites, and disrupted vasculature. The conditional IC-Notch1 expressing transgenic mice provide a unique tool to investigate the Notch pathway using tissue-specific Cre mice and inducible Cre systems.     

Conditional deletion of Notch1 and Notch2 genes in excitatory neurons of postnatal forebrain does not cause neurodegeneration or reduction of Notch mRNAs and proteins

Activation of Notch signaling requires intramembranous cleavage by γ-secretase to release the intracellular domain. We previously demonstrated that presenilin and nicastrin, components of the γ-secretase complex, are required for neuronal survival in the adult cerebral cortex. Here we investigate whether Notch1 and/or Notch2 are functional targets of presenilin/γ-secretase in promoting survival of excitatory neurons in the adult cerebral cortex by generating Notch1, Notch2, and Notch1/Notch2 conditional knock-out (cKO) mice. Unexpectedly, we did not detect any neuronal degeneration in the adult cerebral cortex of these Notch cKO mice up to ～2 years of age, whereas conditional inactivation of presenilin or nicastrin using the same αCaMKII-Cre transgenic mouse caused progressive, striking neuronal loss beginning at 4 months of age. More surprisingly, we failed to detect any reduction of Notch1 and Notch2 mRNAs and proteins in the cerebral cortex of Notch1 and Notch2 cKO mice, respectively, even though Cre-mediated genomic deletion of the floxed Notch1 and Notch2 exons clearly took place in the cerebral cortex of these cKO mice. Furthermore, introduction of Cre recombinase into primary cortical cultures prepared from postnatal floxed Notch1/Notch2 pups, where Notch1 and Notch2 are highly expressed, completely eliminated their expression, indicating that the floxed Notch1 and Notch2 alleles can be efficiently inactivated in the presence of Cre. Together, these results demonstrate that Notch1 and Notch2 are not involved in the age-related neurodegeneration caused by loss of presenilin or γ-secretase and suggest that there is no detectable expression of Notch1 and Notch2 in pyramidal neurons of the adult cerebral cortex.     

Balancer-Cre transgenic mouse germ cells direct the incomplete resolution of a tri-loxP-targeted Cyp1a1 allele, producing a conditional knockout allele

To generate conditional alleles, genes are commonly engineered to contain recognition sites for bacteriophage recombinases, such as Cre recombinase. When such motifs (lox sites) flank essential gene sequences, and provided that Cre recombinase is expressed, Cre recombinase will excise the flanked sequence-creating a conditional knockout allele. Targeted conditional alleles contain a minimum of three lox sites. It would be desirable to have Cre recombinase perform partial resolution (i.e., recombination some of the time between only the two lox sites flanking the marker gene). Here we report use of the commercially available Balancer2-Cre transgenic mouse line to carry out this function from a tri-loxP-site-containing cytochrome p450 1A1 (Cyp1a1) targeted allele. Such incomplete resolution of this complex locus occurred progressively with age in germ cells of male mice; the conditional Cyp1a1 gene was recovered in offspring from mice containing the targeted Cyp1a1 allele and the Cre recombinase transgene. Removal of the marker gene resulted in a conditional Cyp1a1 allele whose expression was indistinguishable from that of the wild-type allele.     

Roles of Pofut1 and O-fucose in mammalian Notch signaling

Mammalian Notch receptors contain 29-36 epidermal growth factor (EGF)-like repeats that may be modified by protein O-fucosyltransferase 1 (Pofut1), an essential component of the canonical Notch signaling pathway. The Drosophila orthologue Ofut1 is proposed to function as both a chaperone required for stable cell surface expression of Notch and a protein O-fucosyltransferase. Here we investigate these dual roles of Pofut1 in relation to endogenous Notch receptors of Chinese hamster ovary and murine embryonic stem (ES) cells. We show that fucosylation-deficient Lec13 Chinese hamster ovary cells have wild type levels of Pofut1 and cell surface Notch receptors. Nevertheless, they have reduced binding of Notch ligands and low levels of Delta1- and Jagged1-induced Notch signaling. Exogenous fucose but not galactose rescues both ligand binding and Notch signaling. Murine ES cells lacking Pofut1 also have wild type levels of cell surface Notch receptors. However, Pofut1-/- ES cells do not bind Notch ligands or exhibit Notch signaling. Although overexpression of fucosyltransferase-defective Pofut1 R245A in Pofut1-/- cells partially rescues ligand binding and Notch signaling, this effect is not specific. The same rescue is achieved by an unrelated, inactive, endoplasmic reticulum glucosidase. Therefore, mammalian Notch receptors require Pofut1 for the generation of optimally functional Notch receptors, but, in contrast to Drosophila, Pofut1 is not required for stable cell surface expression of Notch. Importantly, we also show that, under certain circumstances, mammalian Notch receptors are capable of signaling in the absence of Pofut1 and O-fucose.     

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.     

Notch ligands are substrates for protein O-fucosyltransferase-1 and Fringe

O-Fucose has been identified on epidermal growth factor-like (EGF) repeats of Notch, and elongation of O-fucose has been implicated in the modulation of Notch signaling by Fringe. O-Fucose modifications are also predicted to occur on Notch ligands based on the presence of the C(2)XXGG(S/T)C(3) consensus site (where S/T is the modified amino acid) in a number of the EGF repeats of these proteins. Here we establish that both mammalian and Drosophila Notch ligands are modified with O-fucose glycans, demonstrating that the consensus site was useful for making predictions. The presence of O-fucose on Notch ligands raised the question of whether Fringe, an O-fucose specific beta 1,3-N-acetylglucosaminyltransferase, was capable of modifying O-fucose on the ligands. Indeed, O-fucose on mammalian Delta 1 and Jagged1 can be elongated with Manic Fringe in vivo, and Drosophila Delta and Serrate are substrates for Drosophila Fringe in vitro. These results raise the interesting possibility that alteration of O-fucose glycans on Notch ligands could play a role in the mechanism of Fringe action on Notch signaling. As an initial step to begin addressing the role of the O-fucose glycans on Notch ligands in Notch signaling, a number of mutations in predicted O-fucose glycosylation sites on Drosophila Serrate have been generated. Interestingly, analysis of these mutants has revealed that O-fucose modifications occur on some EGF repeats not predicted by the C(2)XXGGS/TC(3) consensus site. A revised, broad consensus site, C(2)X(3-5)S/TC(3) (where X(3-5) are any 3-5 amino acid residues), is proposed.     

Collagen1alpha1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice

Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse line expressing the Cre recombinase under the control of the rat Collagen1alpha1 (Col1alpha1) promoter (Col1alpha1-Cre). Two founders were identified by genomic PCR from 16 offsprings, and the integration efficiency is 12.5%. In order to determine the tissue distribution and the activity of Cre recombinase in the transgenic mice, the Col1alpha1-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 of Col1alpha1-Cre;Smad4(Co/+)mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon. LacZ staining in the Col1alpha1-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5. Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice. All these data indicated that the Col1alpha1-Cre transgenic mice could serve as a valuable tool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.     

Transvection effects involving DNA methylation during meiosis in the mouse

High efficiencies of recombination between LoxP elements were initially recorded when the Cre recombinase was expressed in meiotic spermatocytes. However, it was unexpectedly found that LoxP recombination fell to very low values at the second generation of mice expressing Cre during meiosis. The inability of the LoxP elements to serve as recombination substrates was correlated with cytosine methylation, initially in LoxP and transgene sequences, but later extending for distances of at least several kilobases into chromosomal sequences. It also affected the allelic locus, implying a transfer of structural information between alleles similar to the transvection phenomenon described in Drosophila. Once initiated following Cre-LoxP interaction, neither cis-extension nor transvection of the methylated state required the continuous expression of Cre, as they occurred both in germinal and somatic cells and in the fraction of the offspring that had not inherited the Sycp1-Cre transgene. Therefore, these processes depend on a physiological mechanism of establishment and extension of an epigenetic state, for which they provide an experimental model.     

Cre activity in fetal albCre mouse hepatocytes: Utility for developmental studies

The albCre transgene, having Cre recombinase driven by the serum albumin (alb) gene promoter, is commonly used to generate adult mice having reliable hepatocyte‐specific recombination of loxP‐flanked (“floxed”) alleles. Based on previous studies, it has been unclear whether albCre transgenes are also reliable in fetal and juvenile mice. Perinatal liver undergoes a dynamic transition from being predominantly hematopoietic to predominantly hepatic. We evaluated Cre activity during this transition in albCre mice using a sensitive two‐color fluorescent reporter system. From fetal through adult stages, in situ patterns of Cre‐dependent recombination of the reporter closely matched expression of endogenous Alb mRNA or protein, indicating most or all hepatocytes, including those in fetal and juvenile livers, had expressed Cre and recombined the reporter. Our results indicate the albCre transgene is effective in converting simple floxed alleles in fetal and neonatal mice and is an appropriate tool for studies on hepatocyte development. genesis 47:789–792, 2009. © 2009 Wiley‐Liss, Inc.     

Hypomorphic phenotype in mice with pituitary‐specific knockout of steroidogenic factor 1

Summary: The bacteriophage Cre recombinase provides a powerful approach for tissue‐specific gene inactivation. Using a Cre transgene driven by the common alpha subunit of glycoprotein hormones (αGSU‐Cre), we have previously inactivated steroidogenic factor 1 (SF‐1) in the anterior pituitary, causing hypogonadotropic hypogonadism with sexual infantilism, sterility, and severe gonadal hypoplasia. We now explore the molecular mechanisms underlying a hypomorphic gonadal phenotype in mice carrying two floxed SF‐1 alleles (F/F) relative to mice carrying one recombined and one floxed allele (F/R). Because their Cre‐mediated disruption of the locus encoding SF‐1 was less efficient, αGSU‐Cre, F/F mice retained some gonadotropin‐expressing cells in the anterior pituitary, thereby stimulating some gonadal function. This novel in vivo model for exploring the effects of differing levels of gonadotropins on gonadal development highlights the need for careful genotype‐phenotype comparisons in studies using Cre recombinase to produce tissue‐specific knockouts. genesis 30:65–69, 2001. © 2001 Wiley‐Liss, Inc.     

Notch signaling regulates late-stage epidermal differentiation and maintains postnatal hair cycle homeostasis

Background

Notch signaling involves ligand-receptor interactions through direct cell-cell contact. Multiple Notch receptors and ligands are expressed in the epidermis and hair follicles during embryonic development and the adult stage. Although Notch signaling plays an important role in regulating differentiation of the epidermis and hair follicles, it remains unclear how Notch signaling participates in late-stage epidermal differentiation and postnatal hair cycle homeostasis.

Methodology and Principal Findings

We applied Cre/loxP system to generate conditional gene targeted mice that allow inactivation of critical components of Notch signaling pathway in the skin. Rbpj, the core component of all four Notch receptors, and Pofut1, an essential factor for ligand-receptor interactions, were inactivated in hair follicle lineages and suprabasal layer of the epidermis using the Tgfb3-Cre mouse line. Rbpj conditional inactivation resulted in granular parakeratosis and reactive epidermal hyperplasia. Pofut1 conditional inactivation led to ultrastructural abnormalities in the granular layer and altered filaggrin processing in the epidermis, suggesting a perturbation of the granular layer differentiation. Disruption of Pofut1 in hair follicle lineages resulted in aberrant telogen morphology, a decrease of bulge stem cell markers, and a concomitant increase of K14-positive keratinocytes in the isthmus of mutant hair follicles. Pofut1-deficent hair follicles displayed a delay in anagen re-entry and dysregulation of proliferation and apoptosis during the hair cycle transition. Moreover, increased DNA double stand breaks were detected in Pofut1-deficent hair follicles, and real time PCR analyses on bulge keratinocytes isolated by FACS revealed an induction of DNA damage response and a paucity of DNA repair machinery in mutant bulge keratinocytes.

Significance

our data reveal a role for Notch signaling in regulating late-stage epidermal differentiation. Notch signaling is required for postnatal hair cycle homeostasis by maintaining proper proliferation and differentiation of hair follicle stem cells.     

Expression and activity of osteoblast-targeted Cre recombinase transgenes in murine skeletal tissues

The Cre/loxP recombination system can be used to circumvent many of the limitations of generalized gene ablation in mice. Here we present the development and characterization of transgenic mice in which Cre recombinase has been targeted to cells of the osteoblast lineage with 2.3 kb (Col 2.3-Cre) and 3.6 kb (Col 3.6-Cre) fragments of the rat Col1a1 promoter. Cre mRNA was detected in calvaria and long bone of adult Col 2.3-Cre and Col 3.6-Cre mice, as well as in tendon and skin of Col 3.6-Cre mice. To obtain a historical marking of the temporal and spatial pattern of Cre-mediated gene rearrangement, Col-Cre mice were bred with ROSA26 (R26R) mice in which Cre-mediated excision of a floxed cassette results in LacZ expression. In Col 2.3-Cre;R26R and Col 3.6-Cre;R26R progeny, calvarial and long bone osteoblasts showed intense beta-gal staining at embryonic day 18 and postnatal day 5. The spatial pattern of beta-gal staining was more restricted in bone and in bone marrow stromal cultures established from Col 2.3-Cre;R26R mice. Similar differences in the spatial patterns of expression were seen in transgenic bone carrying Col1a1-GFP visual reporters. Our data suggest that Col 2.3-Cre and Col 3.6-Cre transgenic mice may be useful for conditional gene targeting in vivo or for obtaining osteoblast populations for in vitro culture in which a gene of interest has been inactivated.