NIEHS Knock Out Core (KOC) Annual Report-2009

National Institute Of Environmental Health Sciences

Investigators

Linked publications, trials & patents

Abstract

The NIEHS knock out core (KOC) is a servicing core facility with some research opportunity. Each project in the core is a long-term commitment from start to finish, and several projects are at different stages of completion in the core at any given time of the year. In Bibliography Section, we are able to list ONLY those articles where core memebers are listed as co-authors but there are many projects or publications that KOC contributed significantly and aren't allowed them to list. Below is the list of projects that were originated and some of the projects that were completed during Oct 2008 to Sep 2009. LIST OF ONGOING PROJECTS IN THE KNOCK OUT CORE (KOC): 1) Analyses of BMP2/4 (KOC/ Dr. Steve Kleeberger): BMP2/4 double hets develop abnormal AV valves (atrioventricular valves between the atria and ventricles that could cause functional abnormalities within the heart. In this on going projects 3-4-months old double hets are implanted with ECG transmitters and the data are collected in hour for 24-hrs. Preliminary results indicate that heart rate and P-wave amplitude are significantly higher in double hets in compare to the age matched controls. 2) Generation of a mouse model with a point mutation in the polymerase domain of DNA pol-beta (Arg to Alanine;R283A) (Drs. Wilson and Asagoshi): DNA polymerase beta is involved in base excision repair pathway in cells and the PI expects that the substitution of Arg283 should develop more carcinogenic phenotype in mice partly due to the accumulation of mutations caused by reduced discriminating ability of the mutated pol-beta. 3) Conditional inactivation of PLCβ1 (Dr. Carmen Williams/LRDT): To prove the hypothesis that egg-derived PLCβ1 is required for calcium oscillatory pattern induced by sperm PLC zeta a floxed PLCβ1 mouse will be generated, and deletion of the gene in the egg in particular will determine the role of this gene and support the hypothesis. homozygotes null mutation exhibit spontaneous seizures and high mortality around 3 weeks of age and never reached the reproductive age. 4) Generation of conditional mice for Limbin (LBN) gene (Dr. Yuji Mishina and KOC): Mutant mice we developed show a high level of postnatal lethality primarily due to an insufficient airway and MRI analysis revealed a collapse trachea with reduced and often-disorganized cartilage. To inactivate the gene in a tissue-specific manner, we propose a conditional LBN mouse model. 5) Generation of CCRP (cytoplasmic CAR retention protein) conditional mouse model (Drs. Negishi and Kanayama): Conditional CCRP KO mice might provide the opportunity to investigate the role of CCRP in regulating the function of the nuclear receptors CAR and PXR at various developmental stages. 6) Generation of Serine 404 Knock-in Mouse in Glucocorticoid Receptor (GR), changing Serine to Alanine (Drs. Cidlowski and Beckley): Dr. Cidlowski's group recently discovered that GR is phosphorylated on a novel site, Serine 404, by Glycogen Synthase Kinase 3-beta (GSK-3b) and prevention of Ser404 phosphorylation enhances GC-dependent transcription and cell death in osteosarcoma cell line. To further explore the significance a knock-in mouse model has been proposed. Current Status: Targeting construct is completed and electroporation is scheduled. 7) Developing a mouse model by deleting tandem repeats in Gnas cluster gene to determine it's role in imprinting (Drs. Birnbaumer and Colaneri): The Gnas cluster genes are located in mouse distal chromosomme 2 and shows a complex imprinting mechanism, and this region is enriched with direct tandem repeat elements. Two mutant mouse models are proposed to determine the role of these tandem repeats. LIST OF PROJECTS THAT ARE COMPLETED DURING THIS PERIOD IN KOC 1) Generation of mutant mice in the Estrogen receptor alpha (ER-Alpha) (Drs. Korach and Arao): Targeting construct having two amino acids altered in exon 9 is completed and introduced a testis-specific promoter, angeotensin-converting enzyme (ACE) promoter driven Cre-recombinase. Mice that are born through germline transmission will have the selection cassette deleted and potentially eliminate the deleterious effect of Neo during development. We successfully generated the mutant animals that are currently under phenotypic analyses. 2) Abnormal Glucose Metabolism in Heterozygous Mutant Mice for A Type I Receptor Required For BMP Signaling (KOC) We analyzed the abnormalities associated with heterozygous mice for Bmpr1a in glucose metabolism during the course of intraperitoneal glucose tolerance test and identified that BMP signaling through BMPRIA plays an important role in glucose metabolism and possibly working through the GSIS pathway 3) Generation of transgenic mice using pre-engineered ES cells containing the mutated loxP site (Dr. Araki and KOC): Dr. Araki, our collaborator in Japan generated several ES cell lines using gene trap. Three transgenic constructs driven by tissue-specific promoters were constructed on the mutant loxP backbone. ES cells harboring the mutant loxP sites were electroporated with these constructs in the presence of a Cre-expressing plasmid and are screened for correct integration of the transgene. The mutant mice we developed using these ES cells are currently under investigation. 4) Generation of Floxed TAB1 mice (TAK1 binding protein) (Drs. Ninomiya-Tsuj/Inagaki/Mishina and KOC): TAB1 knockout mice showed cardiovascular and lung dysmorphogenesis, and died between E15.5 and E18.5. Due to embryonic lethality, it's been difficult to analyze the role of TAB1 in other tissues, like skin homeostasis and we developed TAB1 conditional mouse that would be used for inactivation of the gene using appropriate Cre-expressing mice. 5) Generation of over-expressing inducible Cdx2 mice during development and in adulthood (Drs. Archer/Wang) Cdx are caudle related transcription factors that function redundantly during embryonic development. The Cdx2 homozygote knockout is embryonic lethal before dpc 3.5 because of its involvement in trophectoderm differentiation. The functions of Cdx genes have not been totally understood because of their functional redundancy and early embryonic lethality. To overcome these problems, chimeric mice have been made in the knockout core facility with tetracycline inducible Cdx embryonic stem cells and the function of Cdx genes will be monitored upon Cdx induction during embryonic development as well as in the adulthood. 6) Generation of a mouse line using pre-engineered ES cells containing the mutated loxP site (Drs. Araki (Japan), Mishina and KOC): Modified ES lines from Dr. Araki were injected to determine their ability for germline transmission that we plan to use to introduce transgenic constructs. We have been able to generate these mice using the ES cells we received from our collaborator. 7) We have developed transgenic mouse lines CAG-Z-G11-EGFP for over-expressing G11 using an expression vector that was originally developed in the knock out core. We identified four founder mice that had the ability to produce embryos with LacZ expression in the cranial-facial region before a Cre cross. From those LacZ positive founders, three had visible GFP expression and up-regulated G11 expression, as detected by RT-PCR, after breeding with a Cre reporter mouse.