Embryonic Stem Cell Clones Research Articles

LINE-1 Based Insertional Mutagenesis Screens to Identify Genes Involved in Embryonic Stem Cell Differentiation

Background: Knowledge of the intrinsic properties of embryonic stem cells is essential before the possibility of using these cells for therapeutic purposes becomes a reality. Insertional mutagenesis screens are widely used to identify genes sufficient to confer a particular phenotype; however, applications of such approaches are limited to mouse models. Thus, there is a need to develop a new DNA transposon system that allows gene discovery approaches in stem cells. Methods: This study was performed to explore the possibility of using the long interspersed nuclear element 1 (LINE -1) retrotransposon as a gene trap vector to identify genes involved in the maintenance and differentiation of mouse embryonic stem cells. Results: We developed an episomal, nonviral LINE-1 retrotransposon system using the scaffold/matrix attachment regions in the backbone of our vector. This gene trap vector harbors a GFP marker whose expression occurs only after successful insertional mutagenesis. By utilizing this vector, coupled with GFP expression, we have successfully isolated four individual embryonic stem cell clones that display disrupted genes, including two known genes. We then confirmed the identity of these genes using an inverse PCR approach and verified their function in cell differentiation using shRNAs and undifferentiated markers of embryonic stem cells. Conclusions: The ease of using this insertional mutagen and the simplicity of identifying the cells with disrupted genes by GFP expression make this LINE-1 vector a promising tool for embryonic stem cell and cancer stem cell gene discovery.

Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

Biological Analysis of AML1/RUNX1 Arginine-Mutants by Means of Hematopoietic Rescue Experiments of Runx1-deficient Mouse Embryonic Stem Cells,

Abstract 3382Runt-Related Transcription Factor 1 (RUNX1; also called as Acute Myeloid Leukemia 1: AML1) is one of the most frequently mutated genes associated with human acute leukemia, and encodes DNA binding subunit of the Core-Binding Factor (CBF) transcription complex whose activity is essential for the development of definitive hematopoiesis. RUNX1 serves as a transcriptional activator as well as a repressor to its target genes, depending on the cellular context, mediated through its interaction with co-factors. Increasing evidence obtained these days suggests that post-translational modification of RUNX1, including phosphorylation, methylation, or acetylation on its target amino acid residues, is important for proper and fine tuning of this RUNX1-function, likely by altering its association with functional cofactors. However, biological significance of these modifications has not yet been examined in detail. As an initial effort towards systematic comprehension how these modifications influence RUNX1 function, we tried to evaluate RUNX1 methylation in vitro in this study. Arginine residues just douwnstream to the Runt-domain of RUNX1 were recently reported to be methylated to inhibit corepressor-binding thus enhances its trans-activating activity. In order to elucidate the biological effects of this post-translational modification, we manufactured arginine-to-lysine substitutions at the sites within the mouse cDNA. When these arginine-mutants were exogenously expressed in mammalian cell lines, they showed reduced trans-activating activity detected by a dual-luciferase assay on known reporter constructs in comparison to the wild-type Runx1, confirming previous reports. We then introduced the mutant cDNA into Runx1-deficient mouse embryonic stem (ES) cells by means of a knock-in strategy at the disrupted Runx1 gene locus. These ES cell clones were subjected to the in vitro differentiation to hematopoietic lineages. Wild-type ES cells are known to differentiate into hematopoietic cell lineages via embryoid body formation in a semi-solid culture system, whereas ES cells of Runx1-deficient genotype lose the ability to undergo hematopoietic differentiation. This phenomenon is recognized to be an in vitro phenocopy of the Runx1-deficient mice that suffer from embryonic death due to complete block of fetal liver hematopoiesis. Initial study so far showed that the Runx1-deficient ES cell clones restored the ability to develop hematopoietic cells including macrophages in culture when the arginine-mutant cDNA was re-expressed from the knock-in allele, as is the case for the control Runx1-deficient ES cells with the knocked-in wild-type Runx1. These results suggest that this arginine-to-lysine mutation is dispensable, at least, for the in vitro hematopoietic function of wild-type Runx1 although its trans-activating activity is somewhat impaired. We are currently focusing on introducing this mutation into mouse germ line, and the resultant genome-modified mice should show us the biological significance of the methylation-modification to this important molecule in the context of an entire animal. Disclosures:No relevant conflicts of interest to declare.

Effect of nucleophosmin1 haploinsufficiency on hematopoietic stem cells

Nucleophosmin1 (NPM1), located at 5q35.1, is frequently mutated, deleted, or translocated in a number of hematopoietic malignancies (1). Deletion of 5q encompassing the NPM1 locus occurs in approximately 40% of high risk MDS/AML with complex karyotypes (2), although the common 5q interstitial deletion that occurs in MDS does not include this region (3). Mice genetically engineered to harbor heterozygous loss of Npm1 (Npm1+/−) were shown to have a mild MDS-like phenotype characterized by dyserythropoiesis and dysplastic megakaryocytes with macrocytic anemia, but without significant cytopenias or alterations in lineage commitment. These mice also exhibited increased susceptibility to hematologic malignancies with age (4, 5). While these studies confirmed NPM1’s role as a tumor suppressor, they did not characterize a role for NPM1 in hematopoietic stem or progenitor cell function, a likely phenotype alteration given the propensity of NPM1 mutant mice to develop hematologic neoplasms. In order to better understand the effects of loss of a single NPM1 allele on hematopoiesis, we obtained Npm1 heterozygous deficient mice (#11744-UCD) from the Mutant Mouse Regional Resource Centre (UC Davis, CA). An embryonic stem cell clone with a single gene trap event in the Npm1 locus was used to generate the Npm1+/− line (genetic background 129/SvEvBrd X C57BL/6J).

Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis

Transient receptor potential (TRP) channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca(2+)](i)) through TRPC2 and TRPC3. Because modulation of [Ca(2+)](i) is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild-type littermate controls. Although the erythropoietin-induced increase in [Ca(2+)](i) was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenylhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with a significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. Although erythropoietin-induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis, which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion.

SPOC1 (PHF13) is required for spermatogonial stem cell differentiation and sustained spermatogenesis

SPOC1 (PHF13) is a recently identified protein that has been shown to dynamically associate with somatic chromatin, to modulate chromatin compaction and to be important for proper cell division. Here, we report on the expression of SPOC1 in promyelocytic leukaemia zinc finger (PLZF)-positive undifferentiated spermatogonial stem cells (SSCs) of the mouse testis. To investigate further the biological function of SPOC1 in germ cells we generated Spoc1 mutant mice from a gene-trap embryonic stem cell clone. Postpubertal homozygous Spoc1−/− animals displayed a pronounced progressive loss of germ cells from an initially normal germ epithelium of the testis tubules leading to testis hypoplasia. This loss first affected non-SSC stages of germ cells and then, at a later time point, the undifferentiated spermatogonia. Remarkably, successive loss of all germ cells (at >20 weeks of age) was preceded by a transient increase in the number of undifferentiated Aaligned (Aal) spermatogonia in younger mice (at >10 weeks of age). The number of primary Spoc1−/− gonocytes, the proliferation of germ cells, and the initiation and progression of meiosis was normal, but we noted a significantly elevated level of apoptosis in the Spoc1−/− testis. Taken together, the data argue that SPOC1 is indispensable for stem cell differentiation in the testis and for sustained spermatogenesis.

SPOC1 (PHF13) is required for spermatogonial stem cell differentiation and sustained spermatogenesis

SPOC1 (PHF13) is a recently identified protein that has been shown to dynamically associate with somatic chromatin, to modulate chromatin compaction and to be important for proper cell division. Here, we report on the expression of SPOC1 in promyelocytic leukaemia zinc finger (PLZF)-positive undifferentiated spermatogonial stem cells (SSCs) of the mouse testis. To investigate further the biological function of SPOC1 in germ cells we generated Spoc1 mutant mice from a gene-trap embryonic stem cell clone. Postpubertal homozygous Spoc1(-/-) animals displayed a pronounced progressive loss of germ cells from an initially normal germ epithelium of the testis tubules leading to testis hypoplasia. This loss first affected non-SSC stages of germ cells and then, at a later time point, the undifferentiated spermatogonia. Remarkably, successive loss of all germ cells (at >20 weeks of age) was preceded by a transient increase in the number of undifferentiated A(aligned) (A(al)) spermatogonia in younger mice (at >10 weeks of age). The number of primary Spoc1(-/-) gonocytes, the proliferation of germ cells, and the initiation and progression of meiosis was normal, but we noted a significantly elevated level of apoptosis in the Spoc1(-/-) testis. Taken together, the data argue that SPOC1 is indispensable for stem cell differentiation in the testis and for sustained spermatogenesis.

A resource of vectors and ES cells for targeted deletion of microRNAs in mice

The 21-23 nucleotide single-stranded RNAs classified as microRNAs (miRNA) perform fundamental roles in a wide range of cellular and developmental processes. miRNAs regulate protein expression through sequence-specific base pairing with target messenger RNAs (mRNA) reducing both their stability and the process of protein translation1, 2. At least 30% of protein coding genes appear to be conserved targets for miRNAs1. In contrast to the protein coding genes3, 4, no public resource of miRNA mouse mutant alleles exists. We have generated a library of highly germ-line transmissible C57BL/6N mouse mutant embryonic stem (ES) cells with targeted deletions for the majority of miRNA genes currently annotated within the miRBase registry5. These alleles have been designed to be highly adaptable research tools that can be efficiently altered to create reporter, conditional and other allelic variants. This ES cell resource can be searched electronically and is available from ES cell repositories for distribution to the scientific community6.

Precise BAC targeting of genetically polymorphic mouse ES cells

The use of bacterial artificial chromosomes (BACs) provides a consistent and high targeting efficiency of homologous recombination in embryonic stem (ES) cells, facilitated by long stretches of sequence homology. Here, we introduce a BAC targeting method which employs restriction fragment length polymorphisms (RFLPs) in targeted polymorphic C57BL/6/Cast/Ei F1 mouse ES cell lines to identify properly targeted ES cell clones. We demonstrate that knockout alleles can be generated either by targeting of an RFLP located in the open reading frame thereby disrupting the RFLP and ablating gene function, or by introduction of a transcription stop cassette that prematurely stops transcription of an RFLP located downstream of the stop cassette. With both methods we have generated Rnf12 heterozygous knockout ES cells, which were identified by allele specific PCR using genomic DNA or cDNA as a template. Our results indicate that this novel strategy is efficient and precise, by combining a high targeting efficiency with a convenient PCR based readout and reliable detection of correct targeting events.

Enhanced selection for homologous-recombinant embryonic stem cell clones by Cre recombinase-mediated deletion of the positive selection marker

Enhanced selection for homologous-recombinant embryonic stem cell clones by Cre recombinase-mediated deletion of the positive selection marker

Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice

Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. To evaluate the physiological importance of the enzyme, we generated alk-SMase knockout (KO) mice by the Cre-recombinase-Locus of X-over P1(Cre-LoxP) system and studied SM digestion. Both wild-type (WT) and KO mice were fed ³H-palmitic acid labeled SM together with milk SM by gavage. The lipids in intestinal content, intestinal tissues, serum, and liver were analyzed by TLC. In KO mice, nondigested ³H-SM in the intestinal content increased by 6-fold and the formation of ³H-ceramide decreased markedly, resulting in 98% reduction of ³H-ceramide/³H-SM ratio 1 h after gavage. The absorbed ³H-palmitic acid portion was decreased by 95%. After 3 h, a small increase in ³H-ceramide was identified in distal intestine in KO mice. In feces, ³H-SM was increased by 243% and ceramide decreased by 74% in the KO mice. The KO mice also showed significantly decreased radioactivity in liver and serum. Furthermore, alkaline phosphatase activity in the mucosa was reduced by 50% and histological comparison of two female littermates preliminarily suggested mucosal hypertrophy in KO mice. This study provides definite proof for crucial roles of alk-SMase in SM digestion and points to possible roles in regulating mucosal growth and alkaline phosphatase function.

Deletion of the Distal C Terminus of CaV1.2 Channels Leads to Loss of β-Adrenergic Regulation and Heart Failure in Vivo

L-type calcium currents conducted by CaV1.2 channels initiate excitation-contraction coupling in cardiac and vascular smooth muscle. In the heart, the distal portion of the C terminus (DCT) is proteolytically processed in vivo and serves as a noncovalently associated autoinhibitor of CaV1.2 channel activity. This autoinhibitory complex, with A-kinase anchoring protein-15 (AKAP15) bound to the DCT, is hypothesized to serve as the substrate for β-adrenergic regulation in the fight-or-flight response. Mice expressing CaV1.2 channels with the distal C terminus deleted (DCT-/-) develop cardiac hypertrophy and die prematurely after E15. Cardiac hypertrophy and survival rate were improved by drug treatments that reduce peripheral vascular resistance and hypertension, consistent with the hypothesis that CaV1.2 hyperactivity in vascular smooth muscle causes hypertension, hypertrophy, and premature death. However, in contrast to expectation, L-type Ca2+ currents in cardiac myocytes from DCT-/- mice were dramatically reduced due to decreased cell-surface expression of CaV1.2 protein, and the voltage dependence of activation and the kinetics of inactivation were altered. CaV1.2 channels in DCT-/- myocytes fail to respond to activation of adenylyl cyclase by forskolin, and the localized expression of AKAP15 is reduced. Therefore, we conclude that the DCT of CaV1.2 channels is required in vivo for normal vascular regulation, cell-surface expression of CaV1.2 channels in cardiac myocytes, and β-adrenergic stimulation of L-type Ca2+ currents in the heart.

ELOVL2 controls the level of n-6 28:5 and 30:5 fatty acids in testis, a prerequisite for male fertility and sperm maturation in mice

ELOVL2 is a member of the mammalian microsomal ELOVL fatty acid enzyme family, involved in the elongation of very long-chain fatty acids including PUFAs required for various cellular functions in mammals. Here, we used ELOVL2-ablated (Elovl2(-/-)) mice to show that the PUFAs with 24-30 carbon atoms of the ω-6 family in testis are indispensable for normal sperm formation and fertility in male mice. The lack of Elovl2 was associated with a complete arrest of spermatogenesis, with seminiferous tubules displaying only spermatogonia and primary spermatocytes without further germinal cells. Furthermore, based on acyl-CoA profiling, heterozygous Elovl2(+/-) male mice exhibited haploinsufficiency, with reduced levels of C28:5 and C30:5n-6 PUFAs, which gave rise to impaired formation and function of haploid spermatides. These new insights reveal a novel mechanism involving ELOVL2-derived PUFAs in mammals and previously unrecognized roles for C28 and C30 n-6 PUFAs in male fertility. In accordance with the function suggested for ELOVL2, the Elovl2(-/-) mice show distorted levels of serum C20 and C22 PUFAs from both the n-3 and the n-6 series. However, dietary supplementation with C22:6n-3 could not restore male fertility to Elovl2(+/-) mice, suggesting that the changes in n-6 fatty acid composition seen in the testis of the Elovl2(+/-) mice, cannot be compensated by increased C22:6n-3 content.

A possible role of Reproductive homeobox 6 in primordial germ cell differentiation

Rhox6 is one of the Reproductive Homeobox genes on the X chromosome (Rhox) that is expressed in the placenta and the post-migratory primordial germ cells (PGCs) in the nascent gonad. Despite its novel expression pattern, the significance of Rhox6 expression in the differentiation of these cell types remains unknown. To investigate the role that Rhox6 plays in PGCs, cDNA encoding Rhox6 and short-hairpin (sh) RNA directed against Rhox6 transcripts were introduced by unique expression vectors into a genetically engineered mouse embryonic stem cell (ESC) line. This ESC line expresses enhanced green fluorescent protein (EGFP) under the Oct3/4 promoter, thereby allowing us to monitor the presence of undifferentiated ESCs and PGCs in culture in real time. This ESC line was used to isolate clones that stably expressed Rhox6 cDNA, shRNA against Rhox6 transcripts, or controls. Quantitative RT-PCR results validated that overexpression had been achieved, as well as knockdown of Rhox6 transcripts in these ESC clones. However, these clones exhibited a normal appearance of undifferentiated ESCs and expressed EGFP. Next, these ESC clones were induced to differentiate into PGCs by generating embryoid bodies (EBs) in culture medium without leukemia inhibitory factor. Detection of EGFP expression by fluorescence microscopy and germ cell markers by RT-PCR validated the differentiation of PGCs in EBs. The Rhox6 transgene had little, if any, effect on EGFP expression in EBs, whereas Rhox6 knockdown significantly decreased EGFP expression in EBs. Thus, it is suggested with these results that Rhox6 is necessary for determination of the germ cell lineage.

Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions

Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP–based system, we now report the creation and characterization of a collection of ∼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity.

Spontaneous Irs1 passenger mutation linked to a gene-targeted SerpinB2 allele

In characterizing mice with targeted disruption of the SerpinB2 gene, we observed animals that were small at birth with delayed growth and decreased life expectancy. Although this phenotype cosegregated with homozygosity for the inactive SerpinB2 allele, analysis of homozygous SerpinB2-deficient mice derived from two additional independent embryonic stem (ES) cell clones exhibited no growth abnormalities. Examination of additional progeny from the original SerpinB2-deficient line revealed recombination between the small phenotype (smla) and the SerpinB2 locus. The locus responsible for smla was mapped to a 2.78-Mb interval approximately 30 Mb proximal to SerpinB2, bounded by markers D1Mit382 and D1Mit216. Sequencing of Irs1 identified a nonsense mutation at serine 57 (S57X), resulting in complete loss of IRS1 protein expression. Analysis of ES cell DNA suggests that the S57X Irs1 mutation arose spontaneously in an ES cell subclone during cell culture. Although the smla phenotype is similar to previously reported Irs1 alleles, mice exhibited decreased survival, in contrast to the enhanced longevity reported for IRS1 deficiency generated by gene targeting. This discrepancy could result from differences in strain background, unintended indirect effects of the gene targeting, or the minimal genetic interference of the S57X mutation compared with the conventionally targeted Irs1-KO allele. Spontaneous mutations arising during ES cell culture may be a frequent but underappreciated occurrence. When linked to a targeted allele, such mutations could lead to incorrect assignment of phenotype and may account for a subset of markedly discordant results from experiments independently targeting the same gene.

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca(2+) entry (SOCE) with Orai1 as principal Ca(2+) entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca(2+) entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1(-/-) and Orai1(-/-) platelets had greatly impaired glycoprotein (GP) VI-dependent Ca(2+) signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2(-/-) platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca(2+) signals of Stim1(-/-) and Orai1(-/-) platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1(-/-) and Orai1(-/-) platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, SKF96365, a blocker of (receptor-operated) Ca(2+) entry, inhibited Ca(2+) and procoagulant responses even in Stim1(-/-) and Orai1(-/-) platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca(2+) entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca(2+) entry and PS exposure, only one relying on STIM1-Orai1 interaction.

Gene Correction by Homologous Recombination With Zinc Finger Nucleases in Primary Cells From a Mouse Model of a Generic Recessive Genetic Disease

Zinc Finger nucleases (ZFNs) have been used to create precise genome modifications at frequencies that might be therapeutically useful in gene therapy. We created a mouse model of a generic recessive genetic disease to establish a preclinical system to develop the use of ZFN-mediated gene correction for gene therapy. We knocked a mutated GFP gene into the ROSA26 locus in murine embryonic stem (ES) cells and used these cells to create a transgenic mouse. We used ZFNs to determine the frequency of gene correction by gene targeting in different primary cells from this model. We achieved targeting frequencies from 0.17 to 6% in different cell types, including primary fibroblasts and astrocytes. We demonstrate that ex vivo gene-corrected fibroblasts can be transplanted back into a mouse where they retained the corrected phenotype. In addition, we achieved targeting frequencies of over 1% in ES cells, and the targeted ES cells retained the ability to differentiate into cell types from all three germline lineages. In summary, potentially therapeutically relevant frequencies of ZFN-mediated gene targeting can be achieved in a variety of primary cells and these cells can then be transplanted back into a recipient.

145 PAX2 OVEREXPRESSION IN EMBRYOID BODIES INDUCES UPREGULATION OF INTEGRIN α 8 AND AQUAPORIN-1 INVOLVED IN KIDNEY DEVELOPMENT

You have accessJournal of UrologyStem Cell Research1 Apr 2010145 PAX2 OVEREXPRESSION IN EMBRYOID BODIES INDUCES UPREGULATION OF INTEGRIN α8 AND AQUAPORIN-1 INVOLVED IN KIDNEY DEVELOPMENT Akihiro Nakane, Ryuichi Nishinakamura, Kentaro Mizuno, Yoshiyuki Kojima, Tetsuji Maruyama, Yutaro Hayashi, and Kenjiro Kohri Akihiro NakaneAkihiro Nakane Nagoya, Japan More articles by this author , Ryuichi NishinakamuraRyuichi Nishinakamura Kumamoto, Japan More articles by this author , Kentaro MizunoKentaro Mizuno Nagoya, Japan More articles by this author , Yoshiyuki KojimaYoshiyuki Kojima Nagoya, Japan More articles by this author , Tetsuji MaruyamaTetsuji Maruyama Nagoya, Japan More articles by this author , Yutaro HayashiYutaro Hayashi Nagoya, Japan More articles by this author , and Kenjiro KohriKenjiro Kohri Nagoya, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.198AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Transcription factor Pax2 is essential for kidney development in mice, and overexpression of Pax2 in chick embryos leads to ectopic formation of nephric structures. We have generated embryonic stem (ES) cell lines that repress Pax2 expression in a tetracycline-dependent manner and examined their differentiation potential by embryoid body (EB) formation. METHODS MGZRTcH2 cells, in which gene expression can be controlled by the Tet-off system, were maintained on 0.1% gelatin-coated tissue culture plates in Glasgow minimal essential medium, 10% fetal bovine serum, and 103 units/ml mouse leukemia inhibitory factor at 37°C with 5% CO2. MGZRTcH2 cells were electroporated with the Pax2-containing exchange vector. Eight days after electroporation, Pax2-recombinant clones were picked up and recombination was confirmed by Southern blot analysis. ES cells were cultured in the same medium without LIF to induce EB formation by the hanging drop culture method. EBs were analyzed by reverse transcription-PCR and immunocytochemistry. RESULTS RT-PCR analysis was performed in the parental ES cell line, EBs and clones containing Tet-inducible Pax2. In the presence of Tet (Tet+), gene expression patterns were similar to those in parental cells. In the absence of tetracycline, aquaporin-1 (Aqp1) and integrin α8 were upregulated when cells were induced to form EBs. EBs derived from these cell lines expressed Pax2 and subsequently integrin f¿8 and Aqp1, both of which are possibly involved in kidney development. We further examined expression changes at the protein level. EBs from ES clones containing Tet-inducible Pax2 were cultured with or without tetracycline, and stained with anti-AQP1 and anti-Pax2 antibodies. Pax2 protein was detected only in the absence of Tet. AQP1 protein was also upregulated in the absence of Tet. Considering the slow induction kinetics, our data suggest that Pax2 and additional factors induced in EBs synergistically regulate the two targets. CONCLUSIONS ES cell lines with inducible Pax2 expression will also be useful for dissecting genetic cascades functioning in the development of various organs, including the kidney. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e59 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akihiro Nakane Nagoya, Japan More articles by this author Ryuichi Nishinakamura Kumamoto, Japan More articles by this author Kentaro Mizuno Nagoya, Japan More articles by this author Yoshiyuki Kojima Nagoya, Japan More articles by this author Tetsuji Maruyama Nagoya, Japan More articles by this author Yutaro Hayashi Nagoya, Japan More articles by this author Kenjiro Kohri Nagoya, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The Ubiquitin Ligase Nedd4-1 Is Required for Heart Development and Is a Suppressor of Thrombospondin-1

Nedd4 (Nedd4-1) is a Hect domain E3 ubiquitin ligase that also contains a C2 domain and three WW domains. Despite numerous in vitro studies, its biological function in vivo is not well understood. Here we show that disruption of Nedd4-1 in mice (leaving Nedd4-2 intact) caused embryonic lethality at mid gestation, with pronounced heart defects (double-outlet right ventricle and atrioventricular cushion defects) and vasculature abnormalities. Quantitative mass spectrometry and immunoblot analyses of lysates from the wild type and knock-out mouse embryonic fibroblasts to identify Nedd4-1 in vivo targets revealed dramatically increased amounts of thrombospondin-1 (Tsp-1) in the knock-out mouse embryonic fibroblasts and embryos. Tsp-1 is an inhibitor of angiogenesis, and its elevated level was mediated primarily by enhanced transcription. Interestingly, the administration of aspirin (an inhibitor of Tsp-1) to the pregnant heterozygote mothers led to a reduction in Tsp-1 levels and a substantial rescue of the embryonic lethality. These results suggest that Nedd4-1 is a suppressor of Tsp1 and that increased levels of Tsp-1 in the Nedd4-1 knock-out mice may have contributed to the developmental defect observed in the embryos.