Abstract
We describe a toolkit to perform transgenesis at the ROSA26 locus by recombinase-mediated cassette exchange that will eliminate the inherent problem of random insertion via traditional pronuclear injection. A recombination site-tagged embryonic stem (ES) cell line and two cloning vectors were constructed to facilitate the generation of targeted ES cells with the transgene of interest at ROSA26. The experimental procedure is simple and efficient, and can be readily adapted in many laboratories for rapid generation of transgenic mice.
Highlights
Transgenic mice created by pronuclear injection of a transgene have been a valuable resource for studying gene effects in a whole animal context, albeit there are several pitfalls of this technique
We have developed a toolkit (a ROSA26-tagged mouse embryonic stem (ES) cell line and two exchange vectors) for efficient gene knock-in at the ROSA26 locus in mouse ES cells by recombinase-mediated cassette exchange (RMCE)
We observed that the recombination rate of RMCE was quite high so that recombinants could be isolated
Summary
Transgenic mice created by pronuclear injection of a transgene have been a valuable resource for studying gene effects in a whole animal context, albeit there are several pitfalls of this technique. To circumvent the inherent defect of this type of passive transgenesis, several approaches have been developed[1] that allow transgenes to be inserted at defined genomic loci, such as ROSA26. These active forms of transgenesis can be achieved by utilizing a site-specific recombination system[2] to facilitate the knock-in of the gene of interest. Method First, we generated a mouse ES cell line, designated as R26FNF31F1 (1F1), in which the ROSA26 allele was targeted with a FRT and F3 flanked phosphoglycerate kinase (PGK) promoter and neomycin (Neo) selection cassette by homologous recombination in EC7.1 ES cells (Figure 1A), a hybrid strain with a background of C57BL/6 and 129X1/SvJ3.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
