The core specializes in generating gene targeting and transgenic mouse models. The core has been serving the University of Colorado community since 2005. In that time, we have made over seventy vectors including knock-in, knock-out, conditional knock-out, traditional transgenic, and BAC transgenic. We have also developed specialized vector models for knock-in at the ROSA26 locus. For gene targeting models, our success rate for correct ES cell targeting is 95% and for germline transmission is 100%. The core also offers additional support services such as ES cell screening, BAC purification and mouse genotyping.
Wallace Chick - Co-Director
This vector achieves conditional knock-out of your GOI by deleting a pertinent exon. The exon is floxed and can be deleted by introducing Cre to the system. The vector contains a Neomycin positive selection marker and a TK negative selection marker.
This vector is useful for introducing a mutation to the genome. The mutation could be a base pair change, an amino acid change or fusing the expression of your gene to a marker (ex. Cre). The vector contains a Neomycin positive selection marker and a TK negative selection marker.
Knock-in at the ROSA26 Locus by RMCE
This model is useful for either constitutive or conditional expression of your cDNA at the ROSA26 locus. This method is fast, efficient and results in a more consistent expression of cDNA as compared to traditional transgenic models. Your cDNA can be cloned into one of our pre-designed exchange vectors. The exchange vector is then electroporated into ES cells which already contain the tagged ROSA26 allele. Efficient exchange between the FRT and F3 sited results in incorporation of your cDNA into the ROSA26 locus.
This model achieves a simultaneous deletion of an exon and activation of a marker by utilizing the Flex Switch technique described by Schnutgen et. al. FLEx switch.pdf Initially, the GOI functions normally and no marker is expressed. By utilizing specific recombination sites, loxP and lox511, deletion of exon and activation of marker occurs simultaneously. The expression of the marker can be driven by the endogenous promoter or a strong exogenous promoter (ex. CAG).
The expression of a marker is driven by the promoter of GOI.
The use of BACs has several advantages including the presence of regulatory elements and minimal positional effects. The marker can be of several types including fluorescent tags and Cre derivatives.