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Recombinase
Kinds of Recombinase Terms modified by Recombinase Selected AbstractsExpression of Cre Recombinase in Pigment CellsPIGMENT CELL & MELANOMA RESEARCH, Issue 4 2002Laurence Guyonneau Conditional gene targeting using the Cre/loxP system enables specific deletion of a gene in a tissue of interest. For application of Cre-mediated recombination in pigment cells, Cre expression has to be targeted to pigment cells in transgenic mice. So far, no pigment cell-specific Cre transgenic line has been reported and we present and discuss our first results on use of Cre recombinase in pigment cells. A construct was generated where Cre recombinase is controlled by the promoter of the mouse dopachrome tautomerase (Dct) gene. The construct was functionally tested in vitro and introduced into mice. Following breeding to two reporter mouse strains, we detected Cre recombinase activity in telencephalon, melanoblasts, and retinal pigment epithelium (RPE). Our data demonstrate the feasibility of pigment cell-specific Cre/loxP-mediated recombination. [source] Induction of V(D)J-mediated recombination of an extrachromosomal substrate following exposure to DNA-damaging agentsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 6 2007Robert L. Pinsonneault Abstract V(D)J recombinase normally mediates recombination signal sequence (RSS) directed rearrangements of variable (V), diversity (D), and joining (J) germline gene segments that lead to the generation of diversified T cell receptor or immunoglobulin proteins in lymphoid cells. Of significant clinical importance is that V(D)J-recombinase-mediated rearrangements at immune RSS and nonimmune cryptic RSS (cRSS) have been implicated in the genomic alterations observed in lymphoid malignancies. There is growing evidence that exposure to DNA-damaging agents can increase the frequency of V(D)J-recombinase-mediated rearrangements in vivo in humans. In this study, we investigated the frequency of V(D)J-recombinase-mediated rearrangements of an extrachromosomal V(D)J plasmid substrate following exposure to alkylating agents and ionizing radiation. We observed significant dose- and time-dependent increases in V(D)J recombination frequency (V(D)J RF) following exposure to ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) but not a nonreactive analogue, methylsulfone (MeSulf). We also observed a dose-dependent increase in V(D)J RF when cells were exposed to gamma radiation. The induction of V(D)J rearrangements following exposure to DNA-damaging agents was not associated with an increase in the expression of RAG 1/2 mRNA compared to unexposed controls or an increase in expression of the DNA repair Ku70, Ku80 or Artemis proteins of the nonhomologous end joining pathway. These studies demonstrate that genotoxic alkylating agents and ionizing radiation can induce V(D)J rearrangements through a cellular response that appears to be independent of differential expression of proteins involved with V(D)J recombination. Environ. Mol. Mutagen., 2007. © 2007 Wiley-Liss, Inc. [source] DNA sequence analysis of interlocus recombination between the human T-cell receptor gamma variable (GV) and beta diversity-joining (BD/BJ) sequences on chromosome 7 (inversion 7)ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2 2002Scott W. Ballinger Abstract V(D)J recombinase-mediated recombination between the T-cell receptor (TCR) gamma variable (GV) genes at chromosome 7p15 and the TCR beta joining (BJ) genes at 7q35 leads to the formation of a hybrid TCR gene. These TCR gamma/beta interlocus rearrangements occur at classic V(D)J recombination signal sequences (RSS) and, because the loci are in an inverted orientation, result in inversion events that are detectable in the chromosome structure as inv(7)(p15;q35). Similar rearrangements involving oncogenes and either TCR or immunoglobulin genes mediated by the V(D)J recombinase are found in lymphoid malignancies. Oligonucleotide primers that allow polymerase chain reaction (PCR) amplification across the inv(7) genomic recombination junction sequence have been described. Southern blot analysis has been primarily used to confirm the GV/BJ hybrid nature of the product, with limited information on the DNA sequence of these recombinations. We have modified this PCR method using total genomic DNA from the mononuclear cells in peripheral blood samples to increase specificity and to allow direct sequencing of the translocation junction that results from the recombination between the GV1 and BJ1 families of TCR genes in 25 examples from 11 individuals (three adults, one child, six newborns, and one ataxia telangiectasia (AT) patient). We focused on samples from newborns based on previous studies indicating that the predominant hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutations in newborns are V(D)J recombinase-mediated deletion events and that the frequency of these mutations decreases with increasing age. Although the dilution series-based PCR assay utilized does not yield sharply defined quantitative endpoints, results of this study strongly suggest that inv(7) recombinations in newborns occur at equal or lower frequencies than those seen in adults. Consistent with the PCR primer pairs, all sequenced products contain a GV1 and a BJ1 segment and most also contain a BD1 segment. GV1s2 and 1s4 were the most frequently found GV1 genes (8 and 9 examples, respectively) and BJ1s5 and 1s6 were the most frequently found BJ1 genes (9 and 10 examples, respectively). These results demonstrate the effectiveness of this methodology for assessing GV/BJ interlocus rearrangements mediated by V(D)J recombinase. Environ. Mol. Mutagen. 40:85,92, 2002. © 2002 Wiley-Liss, Inc. [source] Inactivation of the gene for the nuclear receptor tailless in the brain preserving its function in the eyeEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2007Thorsten Belz Abstract During embryogenesis, tailless, an orphan member of the nuclear receptor family, is expressed in the germinal zones of the brain and the developing retina, and is involved in regulating the cell cycle of progenitor cells. Consequently, a deletion of the tailless gene leads to decreased cell number with associated anatomical defects in the limbic system, the cortex and the eye. These structural abnormalities are associated with blindness, increased aggressiveness, poor performance in learning paradigms and reduced anxiousness. In order to assess the contribution of blindness to the behavioural changes, we established tailless mutant mice with intact visual abilities. We generated a mouse line in which the second exon of the tailless gene is flanked by loxP sites and crossed these animals with a transgenic line expressing the Cre recombinase in the neurogenic area of the developing brain, but not in the eye. The resulting animals have anatomically indistinguishable brains compared with tailless germline mutants, but are not blind. They are less anxious and much more aggressive than controls, like tailless germline mutants. In contrast to germline mutants, the conditional mutants are not impaired in fear conditioning. Furthermore, they show good performance in the Morris water-maze despite severely reduced hippocampal structures. Thus, the pathological aggressiveness and reduced anxiety found in tailless germline mutants are due to malformations caused by inactivation of the tailless gene in the brain, but the poor performance of tailless null mice in learning and memory paradigms is dependent on the associated blindness. [source] From meiosis to postmeiotic events: Uncovering the molecular roles of the meiosis-specific recombinase Dmc1FEBS JOURNAL, Issue 3 2010Wataru Kagawa In meiosis, the accurate segregation of maternal and paternal chromosomes is accomplished by homologous recombination. A central player in meiotic recombination is the Dmc1 recombinase, a member of the RecA/Rad51 recombinase superfamily, which is widely conserved from viruses to humans. Dmc1 is a meiosis-specific protein that functions with the ubiquitously expressed homolog, the Rad51 recombinase, which is essential for both mitotic and meiotic recombination. Since its discovery, it has been speculated that Dmc1 is important for unique aspects of meiotic recombination. Understanding the distinctive properties of Dmc1, namely, the features that distinguish it from Rad51, will further clarify the mechanisms of meiotic recombination. Recent structural, biochemical, and genetic findings are now revealing the molecular mechanisms of Dmc1-mediated homologous recombination and its regulation by various recombination mediators. [source] Fast set-up of doxycycline-inducible protein expression in human cell lines with a single plasmid based on Epstein,Barr virus replication and the simple tetracycline repressorFEBS JOURNAL, Issue 3 2007Markus Bach We have developed a novel plasmid vector, pEBTetD, for full establishment of doxycycline-inducible protein expression by just a single transfection. pEBTetD contains an Epstein,Barr virus origin of replication for stable and efficient episomal propagation in human cell lines, a cassette for continuous expression of the simple tetracycline repressor, and a cytomegalovirus-type 2 tetracycline operator (tetO2)-tetO2 promoter. As there is no integration of vector into the genome, clonal isolation of transfected cells is not necessary. Cells are thus ready for use 1 week after transfection; this contrasts with 3,12 weeks for other systems. Adequate regulation of protein expression was accomplished by abrogation of mRNA polyadenylation. In northern analysis of seven cDNAs coding for transport proteins, pools of transfected human embryonic kidney 293 cells showed on/off mRNA ratios in the order of 100 : 1. Cell pools were also analyzed for regulation of protein function. With two transport proteins of the plasma membrane, the on/off activity ratios were 24 : 1 and 34 : 1, respectively. With enhanced green fluorescent protein, a 23 : 1 ratio was observed based on fluorescence intensity data from flow cytometry. The unique advantage of our system rests on the unmodified tetracycline repressor, which is less likely, by relocation upon binding of doxycycline, to cause cellular disturbances than chimera of tetracycline repressor and eukaryotic transactivation domains. Thus, in a comprehensive comparison of on- and off-states, a steady cellular background is provided. Finally, in contrast to a system based on Flp recombinase, the set-up of our system is inherently reliable. [source] Tools for the genetic manipulation of Zygosaccharomyces rouxiiFEMS YEAST RESEARCH, Issue 8 2007Lenka Pribylova Abstract A set of tools for the genetic manipulation of the osmotolerant yeast Zygosaccharomyces rouxii was developed. Auxotrophic mutants (ura3 leu2, ura3 ade2, ura3 leu2 ade2) derived from the CBS 732 type strain were prepared. Centromeric and episomal Z. rouxii/Escherichia coli shuttle plasmids with different marker genes (ScURA3, ZrLEU2, ZrADE2) and with multiple cloning sites were constructed, together with a plasmid enabling green fluorescent protein-tagging. A system for repeatable targeted gene deletion in Z. rouxii was established, involving first the integration of a PCR-generated loxP,kanMX,loxP cassette and second the removal of kanMX from the genome using a Z. rouxii plasmid harbouring cre recombinase. [source] Generation of cortactin floxed mice and cellular analysis of motility in fibroblastsGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 9 2009Shinji Tanaka Abstract Cortactin is an F-actin binding protein that has been suggested to play key roles in various cellular functions. Here, we generated mice carrying floxed alleles of the cortactin (Cttn) gene (Cttnflox/flox mice). Expression of Cre recombinase in mouse embryonic fibroblasts (MEFs) isolated from Cttnflox/flox embryos depleted cortactin within days, without disturbing F-actin distribution and localization of multiple actin-binding proteins. Cre-mediated deletion of Cttn also did not affect cell migration. To obtain mice with a Cttn null allele, we next crossed Cttnflox/flox mice with transgenic mice that express Cre recombinase ubiquitously. Western blot and immunocytochemical analysis confirmed complete elimination of cortactin expression in MEFs carrying homozygously Cttn null alleles. However, we found no marked alteration of F-actin organization and cell migration in Cttn null-MEFs. Thus, our results indicate that depletion of cortactin in MEFs does not profoundly influence actin-dependent cell motility. genesis 47:638,646, 2009. © 2009 Wiley-Liss, Inc. [source] Tissue-specific expression of Cre recombinase from the Tgfb3 locusGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 2 2008Liang-Tung Yang Abstract Tgfb3, a member of the TGF-, superfamily, is tightly regulated, both spatially and temporally, during embryogenesis. Previous mouse knockout studies have demonstrated that Tgfb3 is absolutely required for normal palatal fusion and pulmonary development. We have generated a novel tool to ablate genes in Tgfb3 -expressing cells by targeting the promoterless Cre-pgk-Neo cassette into exon 1 of the mouse Tgfb3 gene, which generates a functionally null Tgfb3 allele. Using the Rosa26 reporter assay, we demonstrate that Cre -induced recombination was already induced at embryonal day 10 (E10) in the ventricular myocardium, limb buds, and otic vesicles. At E14, robust recombination was detected in the prefusion palatal epithelium. Deletion of the TGF-, type I receptor Alk5 (Tgfbr1) specifically in Tgfb3 expressing cells using the Tgfb3-Cre driver line lead to a cleft palate phenotype similar to that seen in conventional Tgfb3 null mutants. In addition, Alk5/ Tgfb3-Cre mice displayed hydrocephalus, and severe intracranial bleeding due to germinal matrix hemorrhage. genesis 46:112,118, 2008. © 2008 Wiley-Liss, Inc. [source] Generation of a transgenic mouse line expressing GFP-Cre protein from a Hoxb4 neural enhancerGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 2 2008Elena Rivkin Abstract Here, we describe a transgenic mouse line, in which expression of green fluorescent protein fused to Cre recombinase (GFP-Cre) is directed by the early neuronal enhancer (ENE) of Hoxb4. In E9.0,13.5 transgenic embryos, Cre activity coincided with endogenous Hoxb4 throughout the neural tube up to the r6/r7 boundary in the hindbrain, the dorsal root ganglia, and the Xth cranial ganglia. Unexpectedly, Cre activity was also consistently detected in the trigeminal (Vth) cranial nerve, which is devoid of endogenous Hoxb4 expression. Strong GFP dependent fluorescence appeared slightly later in E9.5,E11.5 embryos, and reflected the later expression pattern expected for Hoxb4-ENE directed expression in the neural tube up to the r7/r8 not r6/r7 boundary. Thus, with the exception of the trigeminal nerve, this reporter faithfully reproduces endogenous embryonic neural Hoxb4 expression, and provides an excellent reagent for in vivo gene manipulations in neuronal Hoxb4 positive cells as well as the developing trigeminal nerve. This transgenic mouse line should prove especially useful for determining the fate map of neuronal populations arising in rhombomeres 7 and 8 on its own and in combination with the small set of other existing rhombomere-specific Cre recombinase expressing lines. genesis 46:119,124, 2008. © 2008 Wiley-Liss, Inc. [source] Transgenic mouse lines expressing Cre recombinase specifically in posterior notochord and notochord,GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 12 2007Amit Kumar Abstract During development, the organizer provides instructive signals to surrounding cells as well as contributing cells to axial structures. To dissect organizer function at different developmental stages, conditional approaches such as the Cre/loxP system for conditional mutagenesis are particularly useful. Here we describe two new Cre transgenic mouse lines, Foxa2 NFP-Cre and Nodal PNC-Cre, with activity in two organizer domains, the posterior notochord (PNC) and notochord. These lines were made using defined regulatory elements from the Foxa2 and Nodal genes that direct Cre expression in overlapping domains of the PNC and notochord. Our detailed analysis of the timing and location of Foxa2 NFP-Cre and Nodal PNC-Cre activity indicates that these lines are appropriate for conditional mutagenesis of genes expressed from early somite stages onward. genesis 45:729,736, 2007. Published 2007 Wiley-Liss, Inc. [source] Transgenic mice expressing tamoxifen-inducible Cre for somatic gene modification in renal epithelial cellsGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 5 2006Irma S. Lantinga-van Leeuwen Abstract Gene inactivation often leads to an embryonic-lethal phenotype. In focal diseases like renal cell carcinomas and polycystic kidney disease, somatic gene inactivation in subsets of cells is likely to occur at later stages. We generated a transgenic mouse line with an inducible form of Cre recombinase for conditional gene modifications in kidney epithelial cells. To this end a 1.4-kb promoter fragment of the kidney-specific cadherin gene (KspCad) was cloned upstream of a tamoxifen-inducible Cre recombinase (CreERT2) encoding sequence. Expression and activity of Cre was evaluated using reverse transcriptase polymerase chain reaction (RT-PCR) analysis and by crossbreeding to Z/EG reporter mice. One KspCad-CreERT2 line showed kidney-specific Cre expression and mediated recombination upon tamoxifen treatment in Z/EG reporter mice. No reporter gene expression was detected in untreated animals or in extrarenal tissues upon treatment. Within the kidneys, enhanced green fluorescent protein (EGFP) fluorescence was observed in epithelial cells in several nephronic segments. In addition, the system successfully recombined a floxed Pkd1 gene. genesis 44:225,232, 2006. © 2006 Wiley-Liss, Inc. [source] Transgenic expression of Cre recombinase in mitral/tufted cells of the olfactory bulbGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 1 2005Yumiko Nagai Abstract Olfactory information is conveyed from the periphery to the olfactory cortices through mitral and tufted (M/T) cells in the olfactory bulb. A mouse with a specific expression of Cre recombinase in M/T cells is essential for genetic marking of M/T cells and manipulating their properties. Protocadherin 21 (Pcdh21) expression is highly restricted to M/T cells. Here we report a transgenic mouse line, Pcdh21-Cre, in which ,10-kb mouse Pcdh21 promoter drives the expression of Cre recombinase. In Pcdh21-Cre mice, Cre recombinase activity is predominantly detected in M/T cells, visualized with the anti-CFP immunostaining in offspring of a cross between Pcdh21-Cre and the reporter Rosa26-loxP-stop-loxP-CFP strain. These results demonstrate that the ,10-kb Pcdh21 promoter can drive transcription in M/T cells and Pcdh21-Cre mice can be used to excise floxed DNA fragments in M/T cells, which provides a valuable tool to reveal the structure and function of the central olfactory circuits. genesis 43:12,16, 2005. © 2005 Wiley-Liss, Inc. [source] Cre-mediated recombination in cell lineages that express the progesterone receptorGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 2 2005Selma M. Soyal Abstract 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. genesis 41:58,66, 2005. © 2005 Wiley-Liss, Inc. [source] E1-Ngn2/Cre is a new line for regional activation of Cre recombinase in the developing CNSGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 4 2004Joachim Berger Abstract We generated a transgenic mouse line named E1-Ngn2/Cre that expresses Cre recombinase and GFP under the control of the E1 enhancer element of the gene Ngn2 (Scardigli et al.: Neuron 31:203,217, 2001). Cre-recombinase activity and GFP fluorescence are consistent with the reported expression pattern controlled by the E1-Ngn2 enhancer. Recombination was detected in the progenitor domains p1 and p2 in the ventricular zone of the neural tube and in distinct domains of the pretectum, the dorsal and ventral thalamus, the tegmentum of the mesencephalon, and the hindbrain. In the developing cortex, Cre-recombinase activity is confined to a subpopulation of progenitors predominantly in the region of the ventral and lateral pallium. The E1-Ngn2/Cre mouse line thus provides an excellent novel tool for a region-specific conditional mutagenesis in the developing CNS. genesis 40:195,199, 2004. © 2004 Wiley-Liss, Inc. [source] Transgenic expression of Cre recombinase from the tyrosine hydroxylase locusGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 2 2004Jonas Lindeberg Abstract Catecholaminergic neurons are affected in several neurological and psychiatric diseases. Tyrosine hydroxylase (TH) is the first, rate-limiting enzyme in catecholamine synthesis. We report a knockin mouse expressing Cre-recombinase from the 3,-untranslated region of the endogenous Th gene by means of an internal ribosomal entry sequence (IRES). The resulting Cre expression matches the normal pattern of TH expression, while the pattern and level of TH are not altered in the knockin mouse. Crossings with two different LacZ reporter mice demonstrated Cre-mediated genomic recombination in TH expressing tissues. In addition, LacZ was found in some unexpected cell populations (including oocytes), indicating recombination due to transient developmental TH expression. Our novel knockin mouse can be used for generation of tissue-specific or general knockouts (depending on scheme of crossing) in mice carrying genes flanked by loxP sites. This knockin mouse can also be used for tracing cell lineages expressing TH during development. genesis 40:67,73, 2004. © 2004 Wiley-Liss, Inc. [source] Cre-loxP mediated control of PrP to study transmissible spongiform encephalopathy diseasesGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 1 2004Nadia L. Tuzi Abstract Expression of the PrP glycoprotein is essential for the development of the transmissible spongiform encephalopathy (TSE) or prion diseases. Although PrP is widely expressed in the mouse, the precise relevance of different PrP-expressing cell types to disease remains unclear. To address this, we generated two lines of floxed PrP gene-targeted transgenic mice using the Cre recombinase- loxP system. These floxed mice allow a functional PrP allele to be either switched "on" or "off." We demonstrate control of PrP expression for both alleles following Cre-mediated recombination, as determined by PrP mRNA and protein expression in the brain. Moreover, we show that Cre-mediated alteration of PrP expression in these mice has a major influence on the development of TSE disease. These floxed PrP mice will allow the involvement of PrP expression in specific cell types following TSE infection to be defined, which may identify potential sites for therapeutic intervention. genesis 40:1,6, 2004. © 2004 Wiley-Liss, Inc. [source] Generation of a conditional allele of the mouse prostaglandin EP4 receptorGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 1 2004André Schneider Abstract Genetic disruption of the mouse EP4 receptor results in perinatal lethality associated with persistent patent ductus areteriosus (PDA). To circumvent this, an EP4 allele amenable to conditional deletion using the Cre/loxP system was generated. The targeting construct was comprised of a floxed exon2 in tandem with the neomycin-resistance gene in intron 2, flanked by third 3, LoxP site. Mice homozygous for the targeted allele (EP4lox+neo/lox+neo), or following its Cre -mediated deletion (EP4del/del), also die within hours of birth with PDA. In contrast, mice homozygous for a partially recombined allele, retaining exon2 but lacking neo (EP4flox/flox), are viable and show no overt phenotype. Postnatal deletion of the floxed EP4 gene is efficiently achieved in the liver and kidney in a transgenic mouse expressing the inducible Mx1Cre recombinase. The EP mouse should provide a useful reagent with which to examine the physiologic roles of the EP4 receptor. genesis 40:7,14, 2004. © 2004 Wiley-Liss, Inc. [source] Efficient FLP recombination in mouse ES cells and oocytesGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 1 2001Julia Schaft Abstract Summary: We report an improved vector, pCAGGS-FLPe, for transient expression of the enhanced FLP recombinase in mouse ES cells and oocytes. In standard transfection experiments, about 6% of total ES colonies showed FLP recombination, albeit with mosaicism within each colony. After microinjection of pCAGGS-FLPe into oocytes, about one-third of heterozygotic mice born showed complete FLP recombination. Thus pCAGGS-FLPe presents two practical options for removal of FRT cassettes in mice. genesis 31:6,10, 2001. © 2001 Wiley-Liss, Inc. [source] A role for Connexin43 during neurodevelopmentGLIA, Issue 7 2007Amy E. Wiencken-Barger Abstract Connexin43 (Cx43) is the predominant gap junction protein expressed in premitotic radial glial cells and mature astrocytes. It is thought to play a role in many aspects of brain development and physiology, including intercellular communication, the release of neuroactive substances, and neural and glial proliferation and migration. To investigate the role of Cx43 in brain physiology, we generated a conditional knockout (cKO) mouse expressing Cre recombinase driven by the human GFAP promoter and a floxed Cx43 gene. The removal of Cx43 from GFAP-expressing cells affects the behavior of the mice and the development of several brain structures; however, the severity of the phenotype varies depending on the mouse background. One mouse subline, hereafter termed Shuffler, exhibits cellular disorganization of the cortex, hippocampus, and cerebellum, accompanied by ataxia and motor deficits. The Shuffler cerebellum is most affected and displays altered distribution and lamination of glia and neurons suggestive of cell migration defects. In all Shuffler mice by postnatal day two (P2), the hippocampus, cortex, and cerebellum are smaller. Disorganization of the ventricular and subventricular zone of the cortex is also evident. Given that these are sites of early progenitor cell proliferation, we suspect production and migration of neural progenitors may be altered. In conclusion, neurodevelopment of Shuffler/Cx43 cKO mice is abnormal, and the observed cellular phenotype may explain behavioral disturbances seen in these animals as well as in humans carrying Cx43 mutations. © 2007 Wiley-Liss, Inc. [source] Bile duct proliferation in liver-specific Jag1 conditional knockout mice: Effects of gene dosage,HEPATOLOGY, Issue 2 2007Kathleen M. Loomes The Notch signaling pathway is involved in determination of cell fate and control of cell proliferation in multiple organ systems. Jag1 encodes a ligand in the Notch pathway and has been identified as the disease-causing gene for the developmental disorder Alagille syndrome. Evidence from the study of human disease and mouse models has implicated Jag1 as having an important role in the development of bile ducts. We have derived a conditional knockout allele (Jag1loxP) to study the role of Jag1 and Notch signaling in liver and bile duct development. We crossed Jag1loxP mice with a transgenic line carrying Cre recombinase under the control of the albumin promoter and ,-fetoprotein enhancer to ablate Jag1 in hepatoblasts. The liver-specific Jag1 conditional knockout mice showed normal bile duct development. To further decrease Notch pathway function, we crossed the Jag1 conditional knockout mice with mice carrying the hypomorphic Notch2 allele, and bile duct anatomy remained normal. When Jag1 conditional mice were crossed with mice carrying the Jag1 null allele, the adult progeny exhibited striking bile duct proliferation. Conclusion: These results indicate that Notch signaling in the liver is sensitive to Jag1 gene dosage and suggest a role for the Notch pathway in postnatal growth and morphogenesis of bile ducts. (HEPATOLOGY 2007.) [source] The plasticity of immunoglobulin gene systems in evolutionIMMUNOLOGICAL REVIEWS, Issue 1 2006Ellen Hsu Summary:, The mechanism of recombination-activating gene (RAG)-mediated rearrangement exists in all jawed vertebrates, but the organization and structure of immunoglobulin (Ig) genes, as they differ in fish and among fish species, reveal their capability for rapid evolution. In systems where there can exist 100 Ig loci, exon restructuring and sequence changes of the constant regions led to divergence of effector functions. Recombination among these loci created hybrid genes, the strangest of which encode variable (V) regions that function as part of secreted molecules and, as the result of an ancient translocation, are also grafted onto the T-cell receptor. Genomic changes in V-gene structure, created by RAG recombinase acting on germline recombination signal sequences, led variously to the generation of fixed receptor specificities, pseudogene templates for gene conversion, and ultimately to Ig sequences that evolved away from Ig function. The presence of so many Ig loci in fishes raises interesting questions not only as to how their regulation is achieved but also how successive whole-locus duplications are accommodated by a system whose function in other vertebrates is based on clonal antigen receptor expression. [source] In vitro and in vivo studies on the generation of the primary T-cell receptor repertoireIMMUNOLOGICAL REVIEWS, Issue 1 2004Ferenc Livák Summary:, The primary T-cell receptor repertoire is generated by somatic rearrangement of discontinuous gene segments. The shape of the combinatorial repertoire is stereotypical and, in part, evolutionarily conserved among mammals. Rearrangement is initiated by specific interactions between the recombinase and the recombination signals (RSs) that flank the gene segments. Conserved sequence variations in the RS, which modulate its interactions with the recombinase, appear to be a major factor in shaping the primary repertoire. In vitro, biochemical studies have revealed distinct steps in these complex recombinase,RS interactions that may determine the final frequency of gene segment rearrangement. These studies offer a plausible model to explain gene segment selection, but new, more physiological approaches will have to be developed to verify and refine the mechanism by which the recombinase targets the RS in its endogenous chromosomal context in vivo. [source] How to keep V(D)J recombination under controlIMMUNOLOGICAL REVIEWS, Issue 1 2004Marjorie A. Oettinger Summary:, Breaking apart chromosomes is not a matter to be taken lightly. The possible negative outcomes are obvious: loss of information, unstable chromosomes, chromosomal translocations, tumorigenesis, or cell death. Utilizing DNA rearrangement to generate the desired diversity in the antigen receptor loci is a risky business, and it must be carefully controlled. In general, the regulation is so precise that the negative consequences are minimal or not apparent. They are visible only when the process of V(D)J recombination goes awry, as for example in some chromosomal translocations associated with lymphoid tumors. Regulation is imposed not only to prevent the generation of random breaks in the DNA, but also to direct rearrangement to the appropriate locus or subregion of a locus in the appropriate cell at the appropriate time. Antigen receptor rearrangement is regulated essentially at four different levels: expression of the RAG1/2 recombinase, intrinsic biochemical properties of the recombinase and the cleavage reaction, the post-cleavage /DNA repair stage of the process, and accessibility of the substrate to the recombinase. Within each of these broad categories, multiple mechanisms are used to achieve the desired aims. The major focus of this review is on accessibility control and the role of chromatin and nuclear architecture in achieving this regulation, although other issues are touched upon. [source] Positive Regulation of Adult Bone Formation by Osteoblast-Specific Transcription Factor Osterix,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2009Wook-Young Baek Abstract Osterix (Osx) is essential for osteoblast differentiation and bone formation, because mice lacking Osx die within 1 h of birth with a complete absence of intramembranous and endochondral bone formation. Perinatal lethality caused by the disruption of the Osx gene prevents studies of the role of Osx in bones that are growing or already formed. Here, the function of Osx was examined in adult bones using the time- and site-specific Cre/loxP system. Osx was inactivated in all osteoblasts by Col1a1-Cre with the activity of Cre recombinase under the control of the 2.3-kb collagen promoter. Even though no bone defects were observed in newborn mice, Osx inactivation with 2.3-kb Col1a1-Cre exhibited osteopenia phenotypes in growing mice. BMD and bone-forming rate were decreased in lumbar vertebra, and the cortical bone of the long bones was thinner and more porous with reduced bone length. The trabecular bones were increased, but they were immature or premature. The expression of early marker genes for osteoblast differentiation such as Runx2, osteopontin, and alkaline phosphatase was markedly increased, but the late marker gene, osteocalcin, was decreased. However, no functional defects were found in osteoclasts. In summary, Osx inactivation in growing bones delayed osteoblast maturation, causing an accumulation of immature osteoblasts and reducing osteoblast function for bone formation, without apparent defects in bone resorption. These findings suggest a significant role of Osx in positively regulating osteoblast differentiation and bone formation in adult bone. [source] Inactivation of Pten in Osteo-Chondroprogenitor Cells Leads to Epiphyseal Growth Plate Abnormalities and Skeletal Overgrowth,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2007Alice Fiona Ford-Hutchinson Abstract To study the role of the Pten tumor suppressor in skeletogenesis, we generated mice lacking this key phosphatidylinositol 3,-kinase pathway regulator in their osteo-chondroprogenitors. A phenotype of growth plate dysfunction and skeletal overgrowth was observed. Introduction: Skeletogenesis is a complex process relying on a variety of ligands that activate a range of intracellular signal transduction pathways. Although many of these stimuli are known to activate phosphatidylinositol 3,-kinase (PI3K), the function of this pathway during cartilage development remains nebulous. To study the role of PI3K during skeletogenesis, we used mice deficient in a negative regulator of PI3K signaling, the tumor suppressor, Pten. Materials and Methods:Pten gene deletion in osteo-chondrodroprogenitors was obtained by interbreeding mice with loxP-flanked Pten exons with mice expressing the Cre recombinase under the control of the type II collagen gene promoter (Ptenflox/flox:Col2a1Cre mice). Phenotypic analyses included microcomputed tomography and immunohistochemistry techniques. Results: ,CT revealed that Ptenflox/flox:Col2a1Cre mice exhibited both increased skeletal size, particularly of vertebrae, and massive trabeculation accompanied by increased cortical thickness. Primary spongiosa development and perichondrial bone collar formation were prominent in Ptenflox/flox:Col2a1Cre mice, and long bone growth plates were disorganized and showed both matrix overproduction and evidence of accelerated hypertrophic differentiation (indicated by an altered pattern of type X collagen and alkaline phosphatase expression). Consistent with increased PI3K signaling, Pten-deficient chondrocytes showed increased phospho-PKB/Akt and phospho-S6 immunostaining, reflective of increased mTOR and PDK1 activity. Interestingly, no significant change in growth plate proliferation was seen in Pten-deficient mice, and growth plate fusion was found at 6 months. Conclusions: By virtue of its ability to modulate a key signal transduction pathway responsible for integrating multiple stimuli, Pten represents an important regulator of both skeletal size and bone architecture. [source] Osteoblast Deletion of Exon 3 of the Androgen Receptor Gene Results in Trabecular Bone Loss in Adult Male Mice,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2007Amanda J Notini Abstract The mechanism of androgen action on bone was studied in male mice with the AR deleted in mature osteoblasts. These mice had decreased trabecular bone volume associated with a decrease in trabecular number, suggesting that androgens may act directly on osteoblasts to maintain trabecular bone. Introduction: Androgens modulate bone cell activity and are important for the maintenance of bone mass. However, the mechanisms by which they exert these actions on bone remain poorly defined. The aim of this study was to investigate the role of androgens acting through the classical androgen receptor (AR) signaling pathways (i.e., DNA-binding dependent pathways) in osteoblasts using male mice in which exon 3 of the AR gene was deleted specifically in mature osteoblasts. Materials and Methods: Mice with a floxed exon 3 of the AR gene were bred with Col 2.3-cre transgenic mice, in which Cre recombinase is expressed in mineralizing osteoblasts. The skeletal phenotype of mutant mice was assessed by histomorphometry and quantitative ,CT at 6, 12, and 32 weeks of age (n = 8 per group). Wildtype, hemizygous exon 3 floxed and hemizygous Col 2.3-cre male littermates were used as controls. Data were analyzed by one-way ANOVA and Tukey's posthoc test. Results: ,CT analysis of the fifth lumbar vertebral body showed that these mice had reduced trabecular bone volume (p < 0.05) at 32 weeks of age compared with controls. This was associated with a decrease in trabecular number (p < 0.01) at 12 and 32 weeks of age, suggesting increased bone resorption. These effects were accompanied by a reduction in connectivity density (p < 0.01) and an increase in trabecular separation (p < 0.01). A similar pattern of trabecular bone loss was observed in the distal femoral metaphysis at 32 weeks of age. Conclusions: These findings show that inactivation of the DNA binding,dependent functions of the AR, specifically in mature osteoblasts in male mice, results in increased bone resorption and decreased structural integrity of the bone, leading to a reduction in trabecular bone volume at 32 weeks of age. These data provide evidence of a role for androgens in the maintenance of trabecular bone volume directly through DNA binding,dependent actions of the AR in mature osteoblasts. [source] Temporal and Spatial Regulation of CRE Recombinase Expression in Gonadotrophin-Releasing Hormone Neurones in the MouseJOURNAL OF NEUROENDOCRINOLOGY, Issue 7 2008A. Wolfe Gonadotrophin-releasing hormone (GnRH) neurones located within the brain are the final neuroendocrine output regulating the reproductive hormone axis. Their small number and scattered distribution in the hypothalamus make them particularly difficult to study in vivo. The Cre/loxP system is a valuable tool to delete genes in specific cells and tissues. We report the production of two mouse lines that express the CRE bacteriophage recombinase in a GnRH-specific manner. The first line, the GnRH-CRE mouse, contains a transgene in which CRE is under the control of the murine GnRH promoter and targets CRE expression specifically to GnRH neurones in the hypothalamus. The second line, the GnRH-CRETeR mouse, uses the same murine GnRH promoter to target CRE expression to GnRH neurones, but is modified to be constitutively repressed by a tetracycline repressor (TetR) expressed from a downstream tetracycline repressor gene engineered within the transgene. GnRH neurone-specific CRE expression can therefore be induced by treatment with doxycycline which relieves repression by TetR. These GnRH-CRE and GnRH-CRETeR mice can be used to study the function of genes expressed specifically in GnRH neurones. The GnRH-CRETeR mouse can be used to study genes that may have distinct roles in reproductive physiology during the various developmental stages. [source] Altered osteoclast development and function in osteopontin deficient miceJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2008Ahnders Franzén Abstract The role of osteopontin in bone resorption was elucidated by studies of mice with knock out of the osteopontin gene generated by a different approach compared to previous models. Thus, a targeting vector with the promoter region as well as exons 1, 2, and 3 of the osteopontin gene was replaced by a loxP-flanked Neo-TK cassette, and this cassette was eliminated through transient expression of Cre recombinase. The recombined ES cells were used to create mice lacking expression of the osteopontin gene. Tissues from these mice were subjected structural and molecular analyses including morphometry and proteomics. The bone of the null mice contained no osteopontin but showed no significant alterations with regard to other bone proteins. The bone volume was normal in young null animals but in the lower metaphysis, the volume and number of osteoclasts were increased. Notably, the volume and length of the osteoclast ruffled border was several folds lower, indicating a lower resorptive capacity. The null mice did not develop the bone loss characteristic for osteoporosis demonstrated in old wild-type female animals. This quantitative study demonstrates a bone phenotype in the osteopontin null mice of all ages. The data provides further evidence for a role of osteopontin in osteoclast activity. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:721,728, 2008 [source] LeuX tRNA-dependent and -independent mechanisms of Escherichia coli pathogenesis in acute cystitisMOLECULAR MICROBIOLOGY, Issue 1 2008Thomas J. Hannan Summary Uropathogenic Escherichia coli (UPEC) contain multiple horizontally acquired pathogenicity-associated islands (PAI) implicated in the pathogenesis of urinary tract infection. In a murine model of cystitis, type 1 pili-mediated bladder epithelial invasion and intracellular proliferation are key events associated with UPEC virulence. In this study, we examined the mechanisms by which a conserved PAI contributes to UPEC pathogenesis in acute cystitis. In the human UPEC strain UTI89, spontaneous excision of PAI IIUTI89 disrupts the adjacent leuX tRNA locus. Loss of wild-type leuX -encoded tRNA5Leu significantly delayed, but did not eliminate, FimB recombinase-mediated phase variation of type 1 pili. FimX, an additional FimB-like, leuX -independent recombinase, was also found to mediate type 1 pili phase variation. However, whereas FimX activity is relatively slow in vitro, it is rapid in vivo as a non-piliated strain lacking the other fim recombinases rapidly expressed type 1 pili upon experimental infection. Finally, we found that disruption of leuX, but not loss of PAI IIUTI89 genes, reduced bladder epithelial invasion and intracellular proliferation, independent of type 1 piliation. These findings indicate that the predominant mechanism for preservation of PAI IIUTI89 during the establishment of acute cystitis is maintenance of wild-type leuX, and not PAI IIUTI89 gene content. [source] | ||||||||||||||||||||||||||||