NHGRI/DIR Embryonic Stem Cell and Transgenic Mouse Core

作为对NHGRI研究人员的服务，胚胎干细胞和转基因小鼠核心专门从事基因功能和调控的基础研究和人类遗传疾病小鼠模型的创建的转基因小鼠系的产生。核心利用了几种技术来产生转基因小鼠。第一种方法是通过将DNA微注射到受精卵中（原核微注射）来产生种系小鼠。其次，靶向转基因是通过微注射转基因胚胎干细胞（ES细胞）产生的。胚胎干细胞通过在核心基因中同源重组或从其他机构（如IKMC）导入的胚胎干细胞系进行修饰。导入的细胞系需要扩增和存档、DNA/RNA分析、核型和MAP/支原体检测。然后将胚胎干细胞注射到2.5天的8细胞胚胎或3.5天的囊胚中，分别产生完全胚胎干细胞衍生的小鼠或嵌合小鼠。Core还通过将四倍体胚胎与杂交胚胎干细胞聚集在一起产生转基因小鼠。通过使用8细胞或四倍体聚集胚胎产生杂合或完全ES细胞衍生的小鼠，减少了培育嵌合体所需的时间和动物数量。最近，Core已经实现了使用位点特异性核酸酶（CRISPR/Cas和TALENs）一步产生突变小鼠，直接靶向地将DNA和RNA注射到卵原核中。这种直接起始可以产生具有多种基因修饰的种系小鼠。

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