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2004 Scientific Report

Laboratory of Germline

Laboratory of Germline Modification Pamela J. Swiatek, Ph.D. 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. Dr. Swiatek joined VARI as a Special Program Investigator in August 2000. Dr. Swiatek 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. Staff Kellie Sisson, B.S. Julie Koeman, B.S. Juraj Zahatnansky, B.S. Laboratory Members Student Olga Motornao, B.S. Research Interests T he germline modification laboratory is a full-service lab that functions at the levels of service, research, and teaching to develop, analyze, and archive mouse models of human disease. Mouse models are produced using gene targeting technology, a well-established and 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 “knock-in”) or the inactivation of a gene already present 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. VARI and Michigan Life Science Corridor clients are assisted in the design and implementation of gene-targeting experiments and, if necessary, are trained in these techniques. The gene-targeting service has three major parts: DNA electroporation, clone expansion and cryopreservation, and microinjection. Gene targeting procedures are initiated by mutating the genomic DNA of interest and inserting it into embryonic stem (ES) cells using the electroporation technique. The mutated gene integrates into the genome of the ES cell 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 by a process called expansion, 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 called blastocysts. These embryos, containing a mixture of wild-type and mutant ES cells, develop into mice called chimeras. The offspring of chimeras that inherit the mutated gene are called heterozygotes, because they possess one copy of the mutated gene. The heterozygous mice are bred together, or intercrossed, 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 we have the genetically modified mice, our lab assists in developing breeding schemes and provides complete analysis of the mutants. The efficiency of mutant mouse production and analysis is enhanced by the Autogen 9600, a robotic, high-throughput DNA isolation machine. Tail biopsies from genetically engineered mice are processed in a 96-well plate format and the DNA samples are delivered to the client. 42

In addition to the 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 knockouts, 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 what is called 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 ES or fibroblast cell lines from mutant embryos, which allows for in vitro studies of the gene mutation. The germline modification lab has developed, in conjunction with the Laboratory of Cancer Genetics, technical expertise in mouse cytogenetics. Mouse cell lines derived from tumors, fibroblasts, or ES cells can be grown in tissue culture, growth-arrested, fixed, and spread onto glass slides. Spectral karyotyping (SKY) analysis of mouse metaphase chromosome spreads on the slide, using high-quality, 24-color fluorescent in situ hybridization (FISH) paints, can aid in the detection of subtle and complex chromosomal rearrangements. FISH analysis can also be performed on metaphase spreads as described above or on interphase nuclei derived from tissue touch preparations. This type of analysis aids in visualizing specific chromosomal regions of interest. Specialized techniques are available upon request. Finally, the germline modification lab provides cryopreservation services for archiving valuable mouse strains. Mouse embryos or sperm can be cryopreserved in liquid nitrogen and the frozen strains reconstituted using in vitro fertilization techniques. The VARI germline modification lab directs the Michigan Animal Model Consortium (MAMC) of the Core Technology Alliance Corp. This consortium, composed of labs located at VARI and the University of Michigan, receives funding from the Michigan Economic Development Corporation to provide mouse modeling services to Michigan researchers studying human diseases. The services provided include mouse and rat transgenics, TVA transgenics, gene targeting, xenograft models, cytogenetics, embryology, histology, veterinary pathology, sperm and embryo cryopreservation, rederivation, and mouse breeding. These services are described more completely on the MAMC website, . 43

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