As a service to NHGRI investigators, the Embryonic Stem Cell and Transgenic Mouse Core specializes in generating genetically altered mouse lines for basic studies of gene function and regulation and for the creation of mouse models of human genetic diseases. Several technologies are utilized by the Core to generate genetically altered mice. The first method is to create conventional transgenics by microinjection of DNA into fertilized embryos (pronuclear microinjection) to generate germline mice. Secondly, targeted transgenics are generated by microinjecting genetically altered embryonic stem cells (ES cells). The ES cells are modified via homologous recombination of targeted genes in the Core or, are imported ES cell lines from other institutions (i.e., IKMC) Imported lines require expansion and archiving, DNA/RNA analysis, karyotyping, and MAP/Mycoplasma testing. ES cells are then injected into 2.5 day 8-cell embryos or 3.5 day blastocysts to generate fully ES cell-derived or chimeric mice, respectively. The Core also generates transgenic mice by aggregation of tetraploid embryos with hybrid ES cells. By using 8-cell or tetraploid aggregation embryos to produce heterozygous or fully ES cell derived mice, the time and number of animals required is reduced for breeding chimeras. Recently, the Core has implemented the use of site-specific nucleases (CRISPR/Cas and TALENs)to generate mutant mice in one step with direct, targeted injection of DNA and RNA into egg pronuclei. This direct inception can generate germ line mice with multiple gene modifications. The Core archives, in multiple locations, mutant strains by cryopreservation of sperm and embryos and reconstitutes the lines by in vitro fertilization. Thus, the Core can rapidly re-establish mouse strains in the event of a disaster or outbreak as well as easily export lines more efficiently and humanely to other institutions. For quality control, the Core cryopreserves stock embryos from wild-type strains (C57Bl/6J, FVB/N, 129S6Sv/Ev and Balb/c). These embryos are also used for flexible microinjection of 8-cell embryos and fertilized eggs. The use of cryopreserved embryos reduces animal donor needs by 50-60% and allows microinjection of 2-3x more embryos per session. An additional service provided to our institute is to rederive animals into our facility by embryo transfer of fertilized eggs. Fertilized eggs are generated by IVF of imported male sperm with wildtypye oocytes. Any additional eggs/sperm are cryopreserved for disaster. Rederivation generally takes 7-8 weeks as compared to 14-16 weeks for conventional quarantine methods. Closely associated to our rederivation and cryopreservation program, is the in-house breeding colony. This colony rapidly generates mice for experiments and centralizes animals used across animal protocols (ie. cre transgenics). We perform PCR-based genotyping, breed on multiple backgrounds (129 and C57Bl/6) and cryopreserve the lines. We can readily reconstitute mice, which enables reduced rack space for maintenance breeding. Other services include embryo dissection, mouse perfusions, injections, colony maintenance, and animal identification by genotyping. The Core works with NHGRI investigators in construct design, and in basic manipulations of mouse husbandry. The Core is committed to cutting edge transgenic technologies while finding better avenues to reduce the animal requirements. For example, we are investigating inhibitors to improve ES cell quality and improved chimeras. By using inhibitors of GSK3b and MAPK, our parental cell lines and targeted lines have better morphology, controlled growth rate, and higher Nanog levels. This should improve both in-house, and the multiple imported ES cells. The Core has successfully generated mIPS derived stem cells that have been targeted and will transmit through the germ line. We prepare MEFS or tail tip fibroblasts and make them availablee to our investigators who require them for human ES and iPS studies. Moreover, we support characterization of human iPS lines by assisting in the cell injection and generation of teratomas in nude mice. The Core is utilizing protocols for genome editing using TALENs and Crispr/Cas systems. The site specific nuclease systems allows production of genetically engineered mice with multiple gene modifications in a one step process. Core Personnel Description and Equipment Capabilities: The Transgenic Core has seven full-time employees. Lisa Garrett directs and oversees daily operations, training and experimental design. The core staff includes six staff, employed directly by the core, through a branch, or by contract. They include Jun Cheng, Gene Elliott, Kowser Hasneen, Karen Hazzard (all technical staff) and Cecilia Rivas and Elsa Escobar (animal support contractors). The physical organization of the Core is divided into two laboratories behind the animal barrier. The third floor lab houses the central tissue culture space for ES cell growth and maintenance, a cryopreservation area, a microinjection suite with three Zeiss/Eppendorf microinjection stations and one Nikon micromanipulation station, a molecular biology area, and administrative space for 5 individuals. The second floor laboratory is for animal use with downdraft tables and biosafety cabinets. We have a dedicated area for harvesting embryos and tissues with 4 stereomicroscopes that are available to institute investigators. This lab contains a chemical fume hood for ENU, a small animal surgery suite with 3 stereomicroscopes and downdraft tables, a tissue culture area for iPS generation, and ES cell differentiation. There is administrative space for 2 individuals. Summary July 2013-2014 The Core utilized conventional transgenics from 42 DNA constructs( including CRISPR/Cas and TALEN) since July 2013. We have 29 ES cell - targeting constructs that are in various stages of development such as screening for homologous recombination, microinjection, and generation of germline transmitting progeny. I anticipate greater than 20 conventional transgenic constructs for the upcoming year and at least 35 targeting constructs for our institute. During the past year, we rederived in 15 lines of imported mice, cryopreserved 31 lines for disaster preparation, and archived 57 mouse lines. For the upcoming year, we will continue to cryopreserve all mutant mice imported or generated by the Core. I have modified our freezing program to reduce the numbers of embryos frozen (therefore reducing the numbers of mice) and balance the archiving with cryopreserved sperm. We have a significant increase in efficiency of recovering frozen sperm by IVF (>50%) using a modified method by Nakagata et al., J.Mamm.Ova Res. 2010. The Core has generated three new embryonic stem cell lines this past year in addition to our C57Bl6/J and hybrid 129.B6 cell lines. We now have germline competent 129S6 and B6/J albino embryonic stem cells. In addition, we have recently generated C57Bl6/N ES cells and are testing for chimerism and germline transmission. From reviewing Pubmed and information from our investigators, the Core has made substantial contributions on >120 papers from 2005-present. This includes both co-authorship and acknowledgements of several members of the Core. This compilation of papers represents any resource or mouse generated by the Core. Since January 2012, the Core has had co-authorship on at least 4 publications

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National Human Genome Research Institute (NHGRI)
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Khan, Sanjoy Kumar; Yadav, Prem Swaroop; Elliott, Gene et al. (2018) Induced Gnas R201H expression from the endogenous Gnas locus causes fibrous dysplasia by up-regulating Wnt/?-catenin signaling. Proc Natl Acad Sci U S A 115:E418-E427
Zhao, L; Alkadi, H; Kwon, E M et al. (2017) The C-terminal multimerization domain is essential for leukemia development by CBF?-SMMHC in a mouse knockin model. Leukemia 31:2841-2844
Deng, Tao; Postnikov, Yuri; Zhang, Shaofei et al. (2017) Interplay between H1 and HMGN epigenetically regulates OLIG1&2 expression and oligodendrocyte differentiation. Nucleic Acids Res 45:3031-3045
Yang, Wei; Garrett, Lisa; Feng, Di et al. (2017) Wnt-induced Vangl2 phosphorylation is dose-dependently required for planar cell polarity in mammalian development. Cell Res 27:1466-1484
Watkins-Chow, Dawn E; Varshney, Gaurav K; Garrett, Lisa J et al. (2017) Highly Efficient Cpf1-Mediated Gene Targeting in Mice Following High Concentration Pronuclear Injection. G3 (Bethesda) 7:719-722
Crawford, Nicholas G; Kelly, Derek E; Hansen, Matthew E B et al. (2017) Loci associated with skin pigmentation identified in African populations. Science 358:
Chu, Haiyan; McKenna, Mary M; Krump, Nathan A et al. (2016) Reversible binding of hemoglobin to band 3 constitutes the molecular switch that mediates O2 regulation of erythrocyte properties. Blood 128:2708-2716
Huang, Bonnie; Gomez-Rodriguez, Julio; Preite, Silvia et al. (2016) CRISPR-Mediated Triple Knockout of SLAMF1, SLAMF5 and SLAMF6 Supports Positive Signaling Roles in NKT Cell Development. PLoS One 11:e0156072
Meir, Michal; Galanty, Yaron; Kashani, Lior et al. (2015) The COP9 signalosome is vital for timely repair of DNA double-strand breaks. Nucleic Acids Res 43:4517-30
Oliver, Peter L; Chodroff, Rebecca A; Gosal, Amrit et al. (2014) Disruption of Visc-2, a Brain-Expressed Conserved Long Noncoding RNA, Does Not Elicit an Overt Anatomical or Behavioral Phenotype. Cereb Cortex :

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