Mus musculus
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ABSTRACT:
PROVIDER: PRJNA105675 | ENA |
REPOSITORIES: ENA
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Project description:The evolutionarily conserved Delta-Notch cell-cell signal-transduction pathway regulates the determination of various cell fates, which are important for the morphogenesis and development of numerous organs and tissues in many vertebrates and invertebrates. The Dll1 gene was mutated by homologous recombination in embryonic stem cells such that amino acids 2-116 were replaced with an in-frame fusion of the lacZ gene of E.coli. Mouse Dll1 genomic clones were isolated from a strain 129/SvPas library. The replacement vector was constructed by inserting a 4 kb ClaI/XhoI fragment, which encodes a part of the beta-galactosidase gene followed by the PGK-neo cassette, and a 2.6 kb XhoI/EcoRI fragment as the 3M-bM-^@M-^Y homologous region of the replacement vector into pKS+. This vector was linearized with SalI and ClaI, and a 4.1 kb SalI/NcoI as the 5M-bM-^@M-^Y homologous region plus an NcoI/ClaI fragment (800 bp) completing the b-galactosidase gene, were inserted, resulting in the complete replacement vector. This vector was linearized with SalI and electroporated in R1 embryonic stem cells. Correctly targeted clones were identified and verified by Southern blot analysis using external probes from the 3M-bM-^@M-^Y and 5M-bM-^@M-^Y region of the targeted area, and were injected into C57BL/6J embryo to obtain germline transmission. The two analysed Dll1 mutant mouse lines carry the same mutation but are on different genetic background: 1. Heterozygous F1-animals were backcrossed several times to the 129SV/J wild type; 2. Heterozygous F1-animals were outcrossed 11 generation to C3HeB/FeJ wild type Four organs (liver, spleen, thymus, brain) of two Dll1 mutant mouse lines on different genetic background carrying the same mutation were analysed by cDNA microarray technology. Experiment include 4-5 biological replicates for reference (wildtype) and mutant animals. Up to 4 technical replicates for each mutant mouse were performed. As reference pooled RNA was used. 50% of the chip hybridisations are dye sway experiments.
2009-01-01 | E-GEOD-11867 | biostudies-arrayexpress
Project description:BACKGROUND: The Notch signaling pathway is an evolutionary conserved signal transduction pathway involved in embryonic patterning and regulation of cell fates during development and self-renewal. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, involving as well other signal transduction pathways, and implicated in distinct human diseases. Delta-like 1 (Dll1) is one of the known ligands of the Notch receptors. The role of the Notch ligands is less well understood. Loss-of-function of Dll1 leads to embryonic lethality, but reduction of Delta-like 1 protein levels has not been studied in adult stage. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the haploinsufficient phenotype of Dll1 and a missense mutant Dll1 allele (Dll1(C413Y)). Haploinsufficiency leads to a complex phenotype with several biological processes altered. These alterations reveal the importance of Dll1 mainly in metabolism, energy balance and in immunology. The animals are smaller, lighter, with altered fat to lean ratio and have increased blood pressure and a slight bradycardia. The animals have reduced cholesterol and triglyceride levels in blood. At the immunological level a subtle phenotype is observed due to the effect and fine-tuning of the signaling network at the different levels of differentiation, proliferation and function of lymphocytes. Moreover, the importance of the proteolytic regulation of the Notch signaling network emphasized. CONCLUSIONS/SIGNIFICANCE: In conclusion, slight alterations in one player of Notch signaling alter the entire organism, emphasizing the fine-tuning character of this pathway in a high number of processes. Four organs (liver, spleen, thymus, brain) of the Dll1 mutant mouse line analysed by cDNA microarray technology. Experiments include four biological replicates for reference (wildtype) and mutant animals. Two technical replicates for each mutant mouse were performed. As reference pooled RNA of the same organ was used. 50% of the chip hybridisations are dye sway experiments.
2009-03-01 | E-GEOD-12954 | biostudies-arrayexpress
Project description:The evolutionarily conserved Delta-Notch cell-cell signal-transduction pathway regulates the determination of various cell fates, which are important for the morphogenesis and development of numerous organs and tissues in many vertebrates and invertebrates. The Dll1 gene was mutated by homologous recombination in embryonic stem cells such that amino acids 2-116 were replaced with an in-frame fusion of the lacZ gene of E.coli. Mouse Dll1 genomic clones were isolated from a strain 129/SvPas library. The replacement vector was constructed by inserting a 4 kb ClaI/XhoI fragment, which encodes a part of the beta-galactosidase gene followed by the PGK-neo cassette, and a 2.6 kb XhoI/EcoRI fragment as the 3’ homologous region of the replacement vector into pKS+. This vector was linearized with SalI and ClaI, and a 4.1 kb SalI/NcoI as the 5’ homologous region plus an NcoI/ClaI fragment (800 bp) completing the b-galactosidase gene, were inserted, resulting in the complete replacement vector. This vector was linearized with SalI and electroporated in R1 embryonic stem cells. Correctly targeted clones were identified and verified by Southern blot analysis using external probes from the 3’ and 5’ region of the targeted area, and were injected into C57BL/6J embryo to obtain germline transmission. The two analysed Dll1 mutant mouse lines carry the same mutation but are on different genetic background: 1. Heterozygous F1-animals were backcrossed several times to the 129SV/J wild type; 2. Heterozygous F1-animals were outcrossed 11 generation to C3HeB/FeJ wild type
2009-01-01 | GSE11867 | GEO
Project description:Genetic background effect on Scn5a mutant cardiac right ventricle gene expression
| PRJNA122207 | ENA
Project description:BACKGROUND: The Notch signaling pathway is an evolutionary conserved signal transduction pathway involved in embryonic patterning and regulation of cell fates during development and self-renewal. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, involving as well other signal transduction pathways, and implicated in distinct human diseases. Delta-like 1 (Dll1) is one of the known ligands of the Notch receptors. The role of the Notch ligands is less well understood. Loss-of-function of Dll1 leads to embryonic lethality, but reduction of Delta-like 1 protein levels has not been studied in adult stage. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the haploinsufficient phenotype of Dll1 and a missense mutant Dll1 allele (Dll1(C413Y)). Haploinsufficiency leads to a complex phenotype with several biological processes altered. These alterations reveal the importance of Dll1 mainly in metabolism, energy balance and in immunology. The animals are smaller, lighter, with altered fat to lean ratio and have increased blood pressure and a slight bradycardia. The animals have reduced cholesterol and triglyceride levels in blood. At the immunological level a subtle phenotype is observed due to the effect and fine-tuning of the signaling network at the different levels of differentiation, proliferation and function of lymphocytes. Moreover, the importance of the proteolytic regulation of the Notch signaling network emphasized. CONCLUSIONS/SIGNIFICANCE: In conclusion, slight alterations in one player of Notch signaling alter the entire organism, emphasizing the fine-tuning character of this pathway in a high number of processes.
2009-03-01 | GSE12954 | GEO
Project description:Objective DLL1 is a mammalian Notch ligand with essential functions during embryonic development. We tagged endogenous DLL1 in one homologous recombination step such that AcGFP-HA tagged DLL1 could be converted by Cre-mediated site-specific recombination into StrepFlag tagged DLL1. We anticipated that this should allow us to visualise DLL1 in living cells as well as allow for sorting and enrichment of DLL1-expressing cells and efficient purification of DLL1 protein complex. Results We generated constructs to express a DLL1 variant that carried C-terminal in frame an AcGFPHA tag flanked by loxP sites followed by a StrepFlag tag out of frame. Cre-mediated recombination removed AcGFP-HA and added StrepFlag in frame to DLL1. The AcGFPHAstopStrepFlag tag was added to the Dll1 cDNA to allow for tests in cultured cells in vitro and was introduced into endogenous DLL1 in mice using ES cells modified by homologous recombination. Tagged DLL1 protein was detected by antibodies against GFP and HA or Flag, respectively, both in CHO cell and embryo lysates. In CHO cells the AcGFP protein fused to DLL1 was functional. In vivo AcGFP expression was below the level of detection by direct fluorescence. However, the StrepFlag tag allowed us to specifically purify DLL1 complexes from embryo lysates. Homozygous mice expressing AcGFPHA or StrepFlag-tagged DLL1 revealed a vertebral column phenotype reminiscent of disturbances in AP polarity during somitogenesis, a process most sensitive to reduced DLL1 function. Thus, even small C-terminal tags can impinge on sensitive developmental processes requiring DLL1 activity.
2021-11-03 | PXD024680 | Pride
Project description:Production of viable and fertile mice have been reported by nuclear replacement, however, the reasons behind the frequent deformations among foetuses and progeny are not well understood. Activation and in vitro culture methods are also important. Origin of the nuclear donor cells affects success rates, and their survival in culture varies. It has been reported that offspring of nuclear replacement parental mice were normal and fertile. All epigenetic problems in the parents seem to be erased when cell nuclei go through the germ line. cDNA microarray analysis compared expression patterns of Dll1 ko versus Dll1 ko NT (NT = nuclear transfer) and wt versus wt NT of liver and spleen. The aim of this study was analysis of possible epigenetic effects by cloning in the offspring of the 3rd generation. If the 3rd generation cloned animals are indeed phenotypically equivalent to conventional transgenic mouse models then this would open the possibility to develop novel techniques of genetic engineering based on somatic gene targeting and nuclear replacement. four male animals of each cohort (Dll1 conventional transgenic mouse, Dll1 nuclear transfer mouse, wildtype, wildtye nuclear transfer mouse). Two technical replicates including a colour flip experiment. For each analysed organ (liver, spleen) eight experiments including four biological replicates. As reference RNA pools were used
2011-01-01 | E-GEOD-20560 | biostudies-arrayexpress
Project description:Eyeless: Pitx3 mutant mouse line, Expression profiling of liver
| PRJNA116567 | ENA
Project description:The introduction of GFP into the Dll1 locus has made it possible to isolate and study primary intestinal Dll1 positive cells, which constitute the secretory cell precursors of the intestine. Applying DNA array technology, we profiled the RNA changes of FACS-sorted Dll1_high/CD24_low, Dll1_high/CD24_medium and Dll1_high/CD24_high cells . We used cell fractions of intestines from Dll1-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Dll1 promoter. RNA was isolated from three FACS sorted cell populations: all are expressing Dll1-GFP at high levels, but are expressing different levels of Cd24 (low, medium and high). For this set two biological replicates were generated. One additional fraction was isolated from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter. This cell fraction contains intestinal stem cells. Differentially labelled cRNA from these cell fractions was hybridized against a reference (RNA isolated from whole intestine) on 4X44K Agilent Whole Mouse Genome dual colour Microarrays (G4122F).
2012-09-27 | E-GEOD-39915 | biostudies-arrayexpress