As a service to NHGRI investigators, the Transgenic 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. Three separate 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., Bay Genomics). Imported lines require expansion and archiving, DNA/RNA analysis, karyotyping, and MAP/Mycoplasma testing. ES cells are then injected into 2.5 day embryos or 3.5 day blastocysts to generate chimeric mice and then bred to germline transmission. Thirdly, the Core provides a genome-wide approach for our investigators to discover and study new genes by creating mouse mutants through the injection of the chemical mutagen, ENU. Mutagenized mice are bred to assess stability of phenotype and heritability and then transferred to the investigator. Tissues for DNA from mutagenized progeny are archived for mapping and their germplasm cryopreserved. The Core archives all important mutant and transgenic strains by cryopreservation of sperm, embryos, and if necessary, ovaries and re-establishes the lines by in vitro fertilization and embryo transfer. The Core rederives all imported mutant mice for NHGRI investigators into our animal facility by embryo transfer of fertilized eggs. With the availability of cryopreserved embryos and sperm, the Core can readily establish important transgenic mouse strains in the event of a disaster or outbreak as well as easily export lines more efficiently and humanely to other institutions. Other services provided by the Core include embryo dissection, colony maintenance (in house breeding colony of frequently used tester mice for our investigators) and animal identification by genotyping. The Core routinely works with NHGRI investigators in construct design, and in basic manipulations of mouse handling and animal husbandry. Summary The Core generated conventional transgenics from 18 DNA constructs with an average of 8 founder animals per construct in 2009. We anticipate at least 10-20 transgenic constructs per year going forward. We have transfected and/or microinjected over 25 ES cell - targeting constructs that are in various stages of development such as screening for homologous recombination, microinjection, and generation of germline transmitting progeny. We estimate that we will receive greater than 35 targeting constructs this year. To date over 1700 important mutant mouse lines have been cryopreserved in our Core. During the past year, over 75 lines were archived and a reliable and efficient rate of recovery from mutant lines was achieved 10-90% for sperm/IVF (efficiency varies with mutant strains) and 70-90% for embryos. There are approximately 50 strains that are backlogged and we propose to freeze 75-100 new lines per year. The ENU mutagenesis project has been successful with 9 mutants identified from the screening of 701 male G1 progeny. From the inception of the ENU program, a total of 2,136 G1 progeny (male and female) have been produced. Since 2006, 1,363 G1 progeny have been produced with 446 male G1progeny screened and 3 mutants identified. The present objective for this screen is to continue generating mutagenized G1 males that will be bred to sensitized animals for neural crest phenotypes until the gene pool has been saturated and redundant mutations appear. Over 4,000 embryos have been harvested for archiving to detect recessive mutations (G1 x G2). The Core is using inhibitors, GSK3b and MAPK (2i media) in conventional ES growth media. These factors have shown to enhance cell pluripotency/ cell quality and chimera production in our hands. The use of 2i has also enabled the Core to generate ES cells from any mutant mice in more efficient and timely manner. The Core is standardizing protocols for creating induced Pluripotent Stem (iPS) cells from mutant mice. We are using dox-inducible lentiviruses containing the inductive factors. We have generated an iPS clone that shows strong expression of Oct4 and Nanog. Additionally, this iPS clone is karyotypically normal, can differentiate into embryoid bodies and form tumors in nude mice. We are presently microinjecting the iPS clone to generate chimeric animals. Following confirmation of this clone, the Core will provide the service to our investigators to generate iPS cells from transgenic mouse embryonic feeder cells (MEFS), characterize them by immunocytochemistry, real-time PCR and functional analysis such as generating chimeras from these ES-like cells. From reviewing Pubmed and information from our investigators, the Core has made substantial contributions on greater than 100 papers from 2005-2010. 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.
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