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2005 Scientific Report

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Laboratory of Germline

Laboratory of Germline Modification Pamela J. Swiatek, Ph.D., M.B.A. Dr. Swiatek received her M.S. (1984) and Ph.D. (1988) degrees in pathology from Indiana University. From 1988 to 1990, she was a postdoctoral fellow at the Tampa Bay Research Institute. From 1990 to 1994, she was a postdoctoral fellow at the Roche Institute of Molecular Biology in the laboratory of Tom Gridley. From 1994 to 2000, Dr. Swiatek was a Research Scientist and Director of the Transgenic Core Facility at the Wadsworth Center in Albany, N.Y., and an Assistant Professor in the Department of Biomedical Sciences at the State University of New York at Albany. She joined VARI as a Special Program Investigator in August 2000. She has been the chair of the Institutional Animal Care and Use Committee since 2002 and is an Adjunct Assistant Professor in the College of Veterinary Medicine at Michigan State University. Dr. Swiatek received her M.B.A. in 2005 from Krannert School of Management at Purdue University. Staff Julie Koeman, B.S. Kellie Sisson, B.S. Juraj Zahatnansky, B.S. Laboratory Members IACUC Coordinator Kaye Johnson, B.S. Research Interests The germline modification lab is a fullservice lab that functions at the levels of service, research, and teaching to develop, analyze, and maintain mouse models of human disease. Our lab applies a business philosophy to core service offerings and we focus on scientific innovation, customer satisfaction, and service excellence. Mouse models are produced using gene-targeting technology, a wellestablished, powerful method for inserting specific genetic changes into the mouse genome. The resulting mice can be used to study the effects of these changes in the complex biological environment of a living organism. The genetic changes can include the introduction of a gene into a specific site in the genome, (gene “knockin”) or the inactivation of a gene already in the genome (gene “knock-out”). Since these mutations are introduced into the reproductive cells known as the germline, they can be used to study the developmental aspects of gene function associated with inherited genetic diseases. In addition to traditional gene-targeting technologies, the germline modification lab can produce mouse models in which the gene of interest is inactivated in a target organ or cell line instead of in the entire animal. These types of mouse models, known as conditional knock-outs, are particularly useful in studying genes that, if missing, cause the mouse to die as an embryo. The lab also has the capability to produce mutant embryos that have a wild-type placenta using tetraploid embryo technology. This technique is useful when the gene-targeted mutation prevents implantation of the mouse embryo in the uterus. We also assist in the development of embryonic stem (ES) or fibroblast cell lines from mutant embryos, which allows for in vitro studies of the gene mutation. Our gene-targeting service encompasses three major procedures: DNA electroporation, clone expansion and cryopreservation, and microinjection. Gene targeting procedures are initiated by mutating the genomic DNA of interest and inserting it into ES cells using the electroporation technique. The mutated gene integrates into the genome of the ES cells and, by a process called homologous recombination, replaces one of the two wild-type copies of the gene in the cells. Clones are identified, isolated, and cryopreserved, and genomic DNA is extracted from each clone and delivered to the client for analysis. Correctly targeted ES cell clones are thawed, established into tissue culture, and cryopreserved in liquid nitrogen. Gene-targeting mutations are introduced into the mouse by microinjection of the pluripotent ES cell clones into 3.5-day-old mouse embryos (blastocysts). These embryos, containing a mixture of wild-type 42

and mutant ES cells, develop into mice called chimeras. The offspring of chimeras that inherit the mutated gene are heterozygotes, because they possess one copy of the mutated gene. The heterozygous mice are bred together to produce mice that completely lack the normal gene. These homozygous mice have two copies of the mutant gene and are called knock-out mice. Once gene targeting mice are produced, our lab assists in developing breeding schemes and provides for complete analysis of the mutants. The efficiency of mutant mouse production and analysis is enhanced by the AutoGenprep 960, a robotic, high-throughput DNA isolation machine. Tail biopsies from genetically engineered mice are processed in a 96-well format and the DNA samples are delivered to the client for analysis. It is our future plan that the DNA analysis be fully automated, with samples moving directly from the AutoGenprep 960 to a high-throughput genotyping platform, eliminating the need for clients to perform this labor-intensive analysis. The germline modification lab also directs the VARI cytogenetics core, which offers a variety of custom services. Mouse, rat, and human cell lines derived from tumors, fibroblasts, blood, or ES cells can be grown in tissue culture, growtharrested, fixed, and spread onto glass slides. Karyotyping of chromosomes using Leishman- or Giemsa-stained (G-banded) chromosomes is our basic service. However, spectral karyotyping (SKY) analysis of metaphase chromosome spreads, using high-quality, 24-color, wholechromosome fluorescent paints, can aid in the detection of subtle and complex chromosomal rearrangements. Fluorescence in situ hybridization (FISH) analysis, using indirectly or directly labeled bacterial artificial chromosome (BAC) or plasmid probes, can also be performed on metaphase spreads or on interphase nuclei derived from tissue touch preps or nondividing cells. Sequential staining of identical metaphase spreads using FISH and SKY can assist in identifying the chromosome integration site of a randomly integrated transgene. Finally the germline modification lab provides cryopreservation services for archiving and reconstituting valuable mouse strains. These cost-effective procedures decrease the need to continuously breed valuable mouse models, and they provide added insurance against the loss of custom mouse lines due to disease outbreak or a catastrophic event. Eight-cell mouse embryos or mouse sperm can be cryopreserved and stored in liquid nitrogen; they can be reconstituted by implantation into the oviducts of recipient mice or by in vitro fertilization of oocytes, respectively. The VARI germline modification lab directs the Michigan Animal Model Consortium (MAMC) of the Core Technology Alliance (CTA) Corp. MAMC is one of six collaborative core facilities located at the University of Michigan, Michigan State University, Wayne State University, Kalamazoo Community College, and VARI, offering research services in proteomics, structural biology, genomics, bioinformatics, high-throughput compound screening, and animal modeling. These labs receive funding from the Michigan Economic Development Corporation to efficiently provide mouse modeling services to researchers studying human diseases and to promote the commercialization of the core services in order to stimulate the development of biomedical research in Michigan. The MAMC services are classified into three major categories: mouse model development; analysis; and maintenance and preservation. Model development services consist of gene targeting, transgenic, TVA transgenic, and xenotransplantation procedures. Analytical procedures are performed on the animal models to determine the nature and extent of any phenotypic consequences in the models and their correlation with human disease. Mouse analysis consists of histology, necropsy, veterinary pathology, cytogenetics, blood chemistry, blood hematology, and imaging. The DNA isolation service supports the genotyping analysis of the models, and the monoclonal antibody core supports the molecular analysis of the mutant mice and xenotransplantation models. Finally, the maintenance and preservation services include a mouse repository, mouse breeding services, mouse rederivation, and embryo/sperm cryopreservation. These services are described more completely on the MAMC website at . 43

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