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2007 Scientific Report

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VARI | 2007 Cytogenetics Our lab also directs the VARI cytogenetics core, which uses advanced molecular techniques to identify structural and numerical chromosomal aberrations in mouse, rat, and human cells. Tumor, fibroblast, blood, or ES cells can be grown in tissue culture, growth-arrested, fixed, and spread onto glass slides. Karyotyping of chromosomes using Leishman- or Giemsa-stained (G-banded) chromosomes is our basic service; spectral karyotyping (SKY) analysis of metaphase chromosome spreads in 24 colors can aid in detecting 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 help identify the integration site of a randomly integrated transgene. Recently, FISH has been widely used to validate microarray data by confirming amplification/gain or deletion/loss of chromosomal regions of interest. Speed congenics Congenic mouse strain development traditionally involves a series of backcrosses, transferring a targeted mutation or genetic region of interest from a mixed genetic donor background to a defined genetic recipient background (usually an inbred strain). This process requires about ten generations (2.5 to 3 years) to attain 99.9% of the recipient’s genome. Since congenic mice have a more defined genetic background, phenotypic characteristics are less variable and the effects of modifier genes can be more pronounced. Speed congenics, also called marker-assisted breeding, uses DNA markers in a progressive breeding selection to accelerate the congenic process. For high-throughput genotyping, we use the state-of-the-art Sentrix BeadChip technology from Illumina, which contains 1,449 mouse single nucleotide polymorphisms (SNPs). These SNPs are strain-specific and cover the 10 most commonly used inbred mouse strains for optimal marker selection. The client provides the genomic DNA of male mice from the second, third, and fourth backcross generations for genotyping. The males having the highest percentage of the recipient’s genome from each generation are identified, and these mice are bred by the client. Using speed congenics, 99.9% of congenicity can be achieved in five generations (1 to 1.5 years). 53 Michigan Animal Model Consortium The VARI Germline Modification and Cytogenetics lab directs the Michigan Animal Model Consortium (MAMC), one of the ten Core Technology Alliance (CTA) collaborative core facilities located at the University of Michigan, Michigan State University, Wayne State University, Western Michigan University, Kalamazoo Valley Community College, Grand Valley State University, and VARI. The other facilities offer research services in proteomics, bioinformatics, structural biology, genomics, biological imaging, bioscience commercialization, high-throughput compound screening, good manufacturing practices, and antibody technology. The MAMC labs were developed with funding from the Michigan Economic Development Corporation and provide efficient mouse modeling services to researchers studying human diseases. MAMC’s long-term goal is to offer a comprehensive set of cutting-edge services that, through continuous enhancements and development, will define our organization as a single point-of-service site for animal models research. Centralized provision of services maximizes research productivity and decreases time to discovery and is in high demand by academia and pharmaceutical and biotechnology companies, which are increasingly looking to outsource to service centers. Through its well-organized service structure and staff of experts, MAMC supports the growth of the life science industry in Michigan, which is congruent with the CTA goals. From left, front row: Koeman, Sisson, Swiatek, Ayotte back row: Johnson, Lewis, Khoo

Van Andel Research Institute | Scientific Report MAMC service offerings Animal model development • Mouse transgenics. Transgenic technology is used to produce genetically engineered mice expressing foreign genes and serving as models for human disease research. Microinjection delivers the foreign DNA into the pronucleus of a one-cell fertilized egg. This service is provided using various strains of laboratory mice, with production of three transgenic founder mice guaranteed from each procedure. • Gene targeting. By transfecting mouse embryonic stem cells with inactivating, homologous DNAs, target gene expression can be shut down. Genetically engineered mice are produced by microinjecting mutant stem cells into mouse embryos and breeding the progeny to mutant homozygosity. This service is provided using 129 or C57BL/6 embryonic stem cells. • Xenotransplantation. Human cancer cells are injected into immunodeficient mice to produce human-derived tumors. Protocols are designed to test anti-tumor treatment regimens that can lead to prognostic, diagnostic, or therapeutic procedures for humans. Animal model analysis • Cytogenetics. Mouse and rat chromosomal abnormalities and genetic loci are visually observed using Giemsa stain, SKY, or FISH techniques. • Necropsy. Mice are dissected postmortem and tissues are fixed for histological analysis, with necropsy reports generated using voice-recognition software. 54 • Histology. Histological sections are prepared from mouse tissues using microtomes and cryostats; they are processed and stained using automated instruments and then are microscopically analyzed. • Veterinary pathology. A board-certified veterinary pathologist holding the D.V.M. and Ph.D. degrees provides expert microscopic analysis and project consultation. • DNA isolation. DNA is isolated from mouse tail biopsies using the AutogenPrep 960 instrument. Animal model maintenance and preservation • Mouse rederivation. All mouse strains entering the specific pathogen–free breeding facility are rederived to specific pathogen–free mouse status using embryo transfer techniques. • Animal technical services. Veterinary services such as injections, measurements, mating set-up, and tail biopsies are performed by the animal technician staff. • Contract breeding. Wild-type mouse strains and genetically engineered animal models are maintained for research purposes by breeding the strains in a specific pathogen–free environment. • Embryo/sperm cryopreservation. Genetically engineered mice are preserved for archival purposes, disease control, genetic stability, and economic efficiency using germplasm cryopreservation techniques. • Cancer model repository. Mouse cancer models of research interest are maintained through breeding strategies. Recent Publications Furge, Kyle A., Jindong Chen, Julie Koeman, Pamela Swiatek, Karl Dykema, Kseniji Lucin, Richard Kahnoski, Ximing J. Yang, and Bin Tean Teh. 2007. Detection of DNA copy number changes and oncogenic signaling abnormalities from gene expression data reveals MYC activation in high-grade papillary renal cell carcinoma. Cancer Research 67(7): 3171–3176. Zhang, Y.-W., B. Staal, Y. Su, P. Swiatek, P. Zhao, B. Cao, J. Resau, R. Sigler, R. Bronson, and G.F. Vande Woude. 2007. Evidence that MIG-6 is a tumor-suppressor gene. Oncogene 26(2): 269–276. Mukhopadhyay, Rita, Ye-Shih Ho, Pamela J. Swiatek, Barry P. Rosen, and Hiranmoy Bhattacharjee. 2006. Targeted disruption of the mouse Asnal gene results in embryonic lethality. FEBS Letters 580(16): 3889–3894.

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