2008 Scientific Report

VARI | 2008
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.
47