NIEHS Knock Out Core (KOC) Annual Report-2008

Ray, Manas

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 2007 to Sep 2008. ? LIST OF ONGOING PROJECTS IN THE KNOCK OUT CORE (KOC)? 1) 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. Current Statue: Chimeras breeding has been set up for germline transmission ? 2) 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. Current Status: Targeted clones have been identified and submitted to QAL before microinjection. ? 3) 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. Current Status: Targeted clones are amplified and subjected to final Southern analysis before injection. ? 4) 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. Current Status: Targeted clones from each individual construct are currently under expansion for microinjection.? 5) 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.? 6) 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. Current Status: The construct is completed and is ready for electroporation. ? 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. Current Status: Targeting construct has been electroporated into the ES cells and screening is underway. ? 8) Development of FLIP-FLOP System (Dr. Mishina and KOC): By orienting the mutated sites with one site reversed, the recombination event, which would normally cause an excision, will cause an inversion of the contained sequence thus rendering it non-functional. The second recombination event would then invert the previously inverted sequence (thus making it correctly oriented again) and restoring the function of the gene. Current Status: We have generated chimeras and currently breeding to determine the germline transmission. ? ? LIST OF PROJECTS THAT ARE COMPLETED DURING THIS PERIOD IN KOC? 1) Generation of Floxed TAB1 mice (TAK1 binding protein) (Drs. Ninomiya-Tsuj (NC State), 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 is proposed to develop TAB1 conditional mouse. ? 2) Investigate the function of Cdx family members in embryonic hematopoiesis (Drs. Archer and Wang): To investigate if cdx2 plays a role in embryonic hematopoiesis during day 6 to 14.5 in vivo, chimeric analysis has been proposed where cdx2 deficient ESCs will be injected into normal blastocysts, and the embryos from day 6 to day 14.5 will be collected to examine the hematopoietic contribution of cdx2 knockout ESC-derived cells? 3) Knock Out Mouse model for Tbc1d23 gene (Drs. Schwartz and Scott): Tbc1d23 has been identified as a candidate regulator of the response to E. coli or LPS and a knockout mouse model is proposed to determine the in vivo function. ? 4) 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. Several chimeras have been generated and we are breeding them actively for germline transmission. ? 5) Generation of over-expressing inducible Cdx2 mice during development and in adulthood (Drs. Archer and Wang): The functions of Cdx genes have not been totally understood because of their functional redundancy and early embryonic lethality due to Cdx2 deficiency. To overcome these problems, chimeric mice will be made with tetracycline inducible Cdx embryonic stem cells. ? 6) Generation of conditionally over-expressing Type I BMP receptor Alk2 (Ca-Alk2) and Alk6 (Ca-Alk6) mice (Dr. Mishina and KOC): Point mutation in the GS box of type I BMP receptors that alter amino acid Gln to Asp constitutively activate the Ser/Thr kinase without ligands or type II receptor. The expression of mutated cDNA of these type I receptors are driven by a ubiquitous promoter CAG in these transgenic mouse lines.? 7) KO mouse model for Mucine 5ac (Muc5ac) (Drs. Langenbach and Park): The mucins are heavily glycosylated, high molecular weight glycoproteins and inactivation of Muc5ac is proposed to determine its function in lungs susceptibility to infection, gastric ulcer formation and colon cancer. ? 8) Generation of mice lacking proteins involved in the chromatin remodeling process (Drs. Zhang (UNC), Mishina and KOC): It has been evidenced that a protein family containing an ion-binding domain, called JmjC, exhibit a catalytic activity against histone tails, and cause chromatin remodeling. A group of 7-proteins has been selected to develop knockout mouse models that are essential to understand the in vivo function of these proteins. Following members of this family are mutated: Fbxl10; Jmjd1B; Jmjd1A and Jmj