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2009 Scientific Report

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Van Andel Research

Van Andel Research Institute | Scientific Report Research Interests The Germline Modification and Cytogenetics lab is a full-service 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 for both the VARI community and external entities. Our mission is to support mouse model and cytogenetics research with scientific innovation, customer satisfaction, and service excellence. Gene targeting Mouse models are produced using gene-targeting technology, a well-established, 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 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. The germline modification lab can also 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 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 can produce mutant embryos that have a wild-type placenta using tetraploid embryo technology, which 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, to allow 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 is initiated by mutating the genomic DNA of interest and inserting it into ES cells via electroporation. The mutated gene integrates into the genome and, by a process called homologous recombination, replaces one of the two wild-type copies of the gene in the ES 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 by microinjection of the pluripotent ES cell clones into 3.5-day-old mouse embryos (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 heterozygotes possessing one copy of the mutated gene. The heterozygous mice are bred together to produce “knock-out mice” that completely lack the normal gene and have two copies of the mutant gene. Embryo/sperm cryopreservation We provide 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. Mouse embryos at various stages of development, as well as mouse sperm, can be cryopreserved and stored in liquid nitrogen; they can be thawed and used, respectively, by implantation into the oviducts of recipient mice or by in vitro fertilization of oocytes. 44

VARI | 2009 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 (about 1.5 years). Recent Publications Graveel, C.R., J.D. DeGroot, Y. Su, J.M. Koeman, K. Dykema, S. Leung, J. Snider, S.R. Davies, P.J. Swiatek, S. Cottingham, et al. 2009. Met induces diverse breast carcinomas in mice and is associated with human basal breast cancer. Proceedings of the National Academy of Sciences U.S.A. 106(31): 12909–12914. From left: Swiatek, Sisson, Mowry, Lewis, Koeman 45

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