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.
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.
