NHGRI/DIR Embryonic Stem Cell and Transgenic Mouse Core

National Human Genome Research Institute

Investigators

Linked publications, trials & patents

Abstract

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. [unreadable] [unreadable] 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. [unreadable] [unreadable] 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. [unreadable] [unreadable] In addition to the specialized services provided by the Core, we also strive to incorporate new technologies and provide useful tools for our investigators. Recently, we have generated a C57BL/6J ES cell line that has been useful for our investigators that require targeting on this background. We have established MTAs for use of these cells with 14 laboratories (outside of NIH). These labs have reported successful targeting with our C57BL/6J ES cell line. Because of the high targeting efficiency and germline transmission of this useful ES cell line, Core personnel were asked to present at the Transgenic Technology Meeting in Houston, Texas in January 2008. As a result of our report at this meeting, over 20 requests have been initiated for this cell line.[unreadable] [unreadable] We are investigating new areas of assisted reproduction such as Intracytoplasmic Sperm Injection (ICSI) that will aid in the reconstitution of difficult strains. The techniques for ICSI may also be applied to performing nuclear transfer that will be useful to our investigators in future cloning experiments. [unreadable] [unreadable] Summary [unreadable] [unreadable] Since 2004, the Core has generated conventional transgenics from > 82 DNA constructs with an average of 8 founder animals per construct. We anticipate at least 25-30 transgenic constructs per year going forward. We have transfected and/or microinjected over 112 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 1298 important mutant mouse lines have been cryopreserved in our Core. During the past 4 years, over 438 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 15 strains that are backlogged and we propose to freeze 75-100 new lines per year. During 2005-2007, we have imported frozen embryos from 17 mutant lines and have recovered all successfully. From 2005-2007, mice were rescued by in vitro fertilization for 20 out of 23 mutant lines. We have exported over 31 frozen lines (embryos) to collaborating institutes. The Core presented a poster at the 20th International Mammalian Genome Conference in South Carolina in November 2006 on our cryopreservation and reconstitution of sperm from C57Bl6/J and other strains.[unreadable] Since its inception in 2001, the ENU mutagenesis project has been extremely successful with at least 20 mutants identified that are in various stages of mapping, cloning and analysis. The goal of this project is to produce 1000 mutagenized male G1 progeny. To date we have generated 1,501 total G1s (male and female) and have screened (by breeding to a sensitized mutant mouse line) 476/514 G1 males. The recessive screen for the generation of G3 embryos was concluded after > 6000 embryos were analyzed.[unreadable] [unreadable] Other services such as tetraploid aggregation, embryo rederivation, and ES cell importation, testing and karyotyping are performed regularly and offer our investigators efficient means to receive reagents and utilize mice to further their research. Since 2004, we have imported over 58 embryonic stem cell lines and performed successful rederivation by embryo transfer on 53 imported mutant mouse strains.[unreadable] [unreadable] In the past 3 years, the core had the opportunity to lecture eight FAES Biotrac Courses in Transgenic Technologies and Recombinant DNA Methodologies. The director was contacted by Ian Rosewell at Cancer Research in London to serve as an external reviewer for his Transgenic Core. [unreadable] [unreadable] From reviewing Pubmed and information from our investigators, the Core has made substantial contributions on at least 90 papers from 2005-2007. 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.