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Transgenic & Gene Targeting

The goal of the Transgenic and Gene Targeting Core is to provide Wadsworth investigators and their affiliates with centralized access to transgenic technology by serving as a resource for the design, development, analysis and preservation of mutant mouse models. The Transgenic and Gene Targeting Core has provided transgenic services to investigators since 1998 resulting in the production of numerous transgenic and chimeric mouse lines resulting from pronuclear injections and targeted embryonic stem cells. Mice are housed in the AAALAC accredited SPF vivarium. The development of techniques for introducing foreign DNA into the germ line of mice has yielded powerful tools with which to study human diseases and disorders in a physiologically relevant context.

There are primarily two types of transgenic mouse models studied by investigators. The first model is defined by random integration of an exogenously supplied DNA construct. The DNA can be relatively short (5-10kb) in the case of cDNA or very large (150-200kb) such as Bacterial Artificial Chromosomes (BAC). The DNA is introduced via pronuclear injection. Standard pronuclear injection of DNA into fertilized mouse oocytes results in random integration of the transgene into the mouse genome and usually an over expression of the construct. Although cDNA constructs are more efficient than BACs, BACs offer the advantage of harboring endogenous promoters and enhancers and will typically integrate at a lower copy number than cDNA constructs.

The second type of mouse model harbors specific mutations at a genetically defined locus, thus avoiding potential positional effects due to random integration. Targeted modifications can be achieved by first introducing mutations in embryonic stem cells (ESC) via electroporation and homologous recombination of a modified DNA construct. Isolated ESC clones are screened for targeted integration and subsequently injected into mouse embryos. The modified gene remains under endogenous control and is theoretically expressed at endogenous levels. ESC targeting is a lengthy process, however, and the background strain is limited by available germlines transmitting ESC lines. Consequently, an investigator may wish to take advantage of newer transgenic technologies employing targeting nucleases such as CRISPR/Cas9 or TALEN systems. Targeting nucleases can be applied to both knockout and knockin model generation and are not limited by background strain. CRISPR/Cas9 systems are easy to work with, provide specific targeting without overexpression complications and can be accomplished in a fraction of the time compared to ESC based targeting.

In order to request service from the Transgenic and Gene Targeting Core, please contact Dr. Kerri Kluetzman, Director.