Unknown,Transcriptomics,Genomics,Proteomics

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Dll1 mice cloned by nuclear transfer

ABSTRACT: 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

ORGANISM(S): Mus musculus

SUBMITTER: Marion Horsch

PROVIDER: E-GEOD-20560 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

Project description:ADARs, short for adenosine deaminases acting on RNA, were first found by their characteristic activity of deaminating up to 50% of all adenosines in double-stranded RNA in vitro. C. elegans has two ADARs, only one is found in the fly, and mammals express three ADARs. Importantly, the spectrum of physiological roles of these enzymes in the different organisms remains largely unknown. ADARs are ~750-1150 amino acids in size, feature 2-3 N-terminal double-stranded RNA binding motifs, an enzymatic domain related to that of prokaryotic cytidine deaminases, and a ~190 residue extended C-terminal domain of unknown function. The mammalian ADARs are detectable in many if not all tissues, with relatively prominent expression in the central nervous system, predicting tissue- or cell type-specific tasks for the mammalian ADARs. Studies over the last decade collectively indicated that one function of ADARs is the editing of nuclear transcripts to introduce select amino acid substitutions in proteins. Arguably the best-studied example for this type of RNA editing (‘A-to-I editing’) in the mammal is performed by ADAR2 and occurs in primary transcripts for the GluA2 subunit (also known as GluR-B or GluR2 subunit) of AMPA receptors mediating fast excitatory neurotransmission. A particular glutamine codon (CAG) in the exonic sequence for the inner lining of the glutamate-activated AMPA receptor is converted by deamination of the central adenosine into an unusual arginine codon (CIG; the resultant inosine is seen as guanosine by the protein translation machinery). This substitution of the gene-encoded glutamine codon by the edited arginine codon is found in nearly 100% of all GluA2 cDNA derived from brain mRNA. RNA editing in this instance ensures that GluA2-containing AMPA receptors become impermeable to Ca2+. In addition to switching a key functional determinant in GluA2 to nearly 100%, ADAR2 has been implicated in generating numerous amino acid substitutions at lower levels in various mammalian neurotransmitter receptors and voltage-gated ion channels, primarily but not exclusively of nervous tissue. Most of these substitutions elicit distinct functional consequences, which led to the notion that site-selective RNA editing by ADAR2 may ensure the functional fine-tuning of the affected transmembrane proteins. Such fine-tuning should reflect the expansion of the protein sequence directed by the respective gene into sequence subpopulations, each differing in particular functional aspects, such as transmitter efficacy, desensitization, or voltage threshold. If this notion is correct, ADAR2 knockout should elicit phenotypes, which might range from subtle to severe, depending on the physiological role(s) of the ADAR2 targets in different tissues. ADAR2 knockout mice, however, die soon after birth from severe epileptic seizures, a consequence of the changed ion conductance properties of AMPA receptors containing unedited GluA2. Fortunately, mice lacking ADAR2 are robust and have normal life span if they carry GluA2 alleles genetically altered to contain the particular arginine codon for a Ca2+ impermeable ion channel pore of AMPA receptors, thus rescinding the need for ADAR2-mediated GluA2 transcript editing. These mice are thus eminently suitable to study the phenotypic consequences of failure to edit all ADAR2 targets excepting GluA2. We therefore tested cohorts of ADAR2-/-/GluA2(R)/(R) mice in 16 well-established paradigms for central and peripheral phenotypic abnormalities. We report that we found few differences to control mice (GluA2(R)/(R)) and hence, the lack of ADAR2 appears not to affect most of the physiological functions tested. Brain of four mutant animals of ADAR-/-/GluR-BR/R versus a pool of four reference (wildtype) mice; two technical replicates for each mutant mouse including a dye swap experiment - in total 8 chip hybridisations

Project description:The alpha-fetoprotein (AFP) gene is a member of the albumin gene family and encodes a serum glycoprotein with an onco-fetal pattern of expression. AFP is produced in high concentrations during embryonic life by the hepatocytes and the visceral endoderm of the yolk sac and to a lesser extent by the developing gastrointestinal tract and kidney. Alpha-fetoprotein is the major serum fetal protein in mammals, with a concentration that reaches the order of several mg/ml. Its synthesis decreases dramatically shortly after birth to reach only trace amounts a few weeks later but can be restored during life when liver pathologies or some types of tumors develop (hepatitis, cirrhosis, hepatoma, teratocarcinoma, and some pancreatic and renal tumors). AFP is then mainly expressed by the adult liver. AFP produced by the embryo is secreted in the amniotic fluid and is able to cross the placental barrier to reach the maternal blood circulation, where its titer is used as a diagnostic marker to reveal developmental anomalies of the fetus. Abnormally high levels of AFP in the maternal serum indicates elevated risk for neural tube defects of the fetus such as spina bifida or anencephaly, whereas abnormally low levels indicates elevated risk for a Down’s syndrome. Measurements of the AFP levels in the maternal serum are undertaken at 14-22 weeks of each pregnancy and are part, along with unconjugated estradiol, human chorionic gonadotropin and inhibin A, of the quadruple test for antenatal Down’s syndrome screening. Abnormal AFP levels can also be indicative of other fetal pathologies. The reason why these pathologies induce abnormal levels of AFP is still not known. Homozygous Afp knock-out females (AFP KO) are sterile, due to anovulation. Ovaries contain mature follicles and are functional, but lack a proper signal from the hypothalamic-pituitarian axis (HPG) in order to ovulate. The expression profile of several genes in the HPG is impaired. Furthermore, AFP KO females are masculinized and defeminized. They exhibit a loss of female sexual behaviour (no lordosis) and a male pattern of tyrosine hydroxylase expressing neurons distribution in some sexual dimorphic areas of the brain. The phenotype of those mice is a consequence of an estrogen overexposure of their brains during the perinatal period. This phenotype can be reversed if these mice develop in an embryonic environment strongly reduced in estrogens. Homozygous AFP KO mice that developed in those conditions are fertile in adulthood (no further treatment needed), and exhibit proper sexual behaviour. cDNA microarray analysis of embryonic and adult liver tissue of AFP1 kO mice. Single mutant mice were analysed versus a pool of wt RNA in two technical replicates (only for adult tissue) including a dye swap experiment. In total 6 adult mutant male mice and 6 embryos (3 male and 3 female) were analysed

Project description:Col1a1 is located on mouse distal chromosome 11 (Celera 94607393-94622990bp; 15kb) and encodes type I procollagen that associates stoichiometrically with col1a2 in forming secreted type I mature collagen that self-assembles into a supramolecular fibrillar structure consisting of a protein family. Type I collagens are predominantly enriched in bone, cartilage, skin, tendons, and eye (i.e. in major fibrous tissues) making up the main extracellular matrix (ECM) component for support and scaffolding purposes. Col1a1 is known to be expressed in the embryo (E9.5; Northern dot blot), perioptic mesenchyme (E11), cornea (E11), skeleton (E14.5), brain meninges (E14.5), and cartilage (E14.5) at relatively early stages. Most evidence of type I collagen function is coupled to the latter stages of skeletal development, thus bypassing the major events of axis formation, tissue differentiation, and mesenchlymal-epithial interactions, for example, that are more symbolic of morphogens and growth factors. Members of the integrin cell surface receptor family of molecules mediate cell adhesion to ECM proteins such as collagen (mainly to type IV basement membrane collagen that forms into a polygonal meshwork), fibronectin, and laminin implicated in wound healing and inflammatory responses (e.g. Crohn disease, ulcerative colitis) in connective tissues. There is some evidence for protein-protein interactions between leukocyte-associated integrins and the interstitial matrix in promoting the migration and/or activation of extravasated leukocytes (e.g., T cells and monocytes) within the perivascular compartment, an ECM-rich environment. This region is surmised to be an important location where certain human leukocytes undergo differentiation (e.g. monocytes) and activation (neutrophils, monocytes, lymphocytes) upon extravascular migration. The von Willebrand factor (vWF), type C motif is found in various plasma proteins like complement factors, the integrins, collagen types, and other extracellular proteins. Thus, the majority of vWF-containing proteins are extracellular and a common feature appears to be involvement in multi protein complexes participating in numerous biological events (e.g. cell adhesion, migration, homing, pattern formation, and signal transduction). Three organs (lung, heart and bone) of 5 AGA002 mutant mice (age 2x 10 days and 3x 11 days) were analysed by cDNA microarray technology. As reference five mice, 11 days old were used 50% of the chip hybridisations are dye swap experiments.

Project description:Background: Epithelial membrane protein 3 (EMP3) was previously reported to be involved in immunological reactions and in tumor as potential suppressor, as well as a candidate gene for cardiac dystrophic calcification (DCC). Given its assumed role in different disorders, we aimed to generate and perform a generalized phenotypic screening of EMP3-knockout mice. Materials and Methods: TaconicArtemis used knockout-targeting strategy and generated knockout mice for EMP3. Large-scale phenotype screens were performed at the German Mouse Clinic (GMC) to obtain a standardized and comprehensive way of phenotype. The phenotype screens involved tests for nearly 320 parameters in different screening areas such as: dysmorphology, behavior, neurology, eye, nociception, energy metabolism, clinical chemistry and hematology, immunology, allergy, steroid metabolism, cardiovascular and lung function and pathology. For DCC, screening was performed in our laboratory using the freeze-thaw injury method. Results: Here, we report on the phenotype of EMP3 knockout. EMP3-KO mice are viable and fertile. Under baseline conditions, an aberrant immunological phenotype was found with nearly 70% penetrance in mutant male mice. Specifically, a lower frequency of T cells and an inverse trend in B cells was observed. Also a higher frequency of B cells was seen in female mutant mice. For all the remaining screening, no genotype-specific differences were found. Also no calcification deposits were found in the EMP3-KO mice as response to injury. In addition we reviewed and discussed the strong role of EMP3 in apoptosis and tumor. Therefore, further analysis of EMP3 as tumor suppressor gene in EMP3-KO mice required investigation under challenging conditions. Conclusion: Large-scale phenotypic screening suggests a role of EMP3 under basal conditions in immunity. Differences in the proportion of various leukocyte subsets from the corresponding wild type were found. Further investigation is ongoing to demonstrate the role of EMP3 as tumor suppressor in a sensitized model. Finally, we excluded the EMP3 gene as candidate gene for DCC on the C57BL/6 genetic background. Thymus and brain of four mutant animals of the EMP3 mutant mice versus a pool of four reference (wildtype) mice; two technical replicates for each mutant mouse including a dye swap experiment - in total 8 chip hybridisations for each of the analysed organs

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.

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Dll1 mice cloned by nuclear transfer

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