Targeting constructs were generated using a combined gene synthesis (GenScript Corp.) and molecular cloning approach. Briefly, to target the Rosa26 locus, a cassette containing the following components was constructed: FRT – LoxP – Stop codons – 3x SV40 polyA – LoxP – EYFP – WPRE – bGH polyA – AttB – PGK promoter – FRT – Neo – PGK polyA – AttP. For most targeting vectors, this cassette was cloned into a Rosa-CAG targeting vector3 (link), downstream of the CAG promoter and upstream of the 3′ arm, to generate the final EYFP targeting vector. Unique restriction sites flanking the EYFP gene were used to replace EYFP with alternative reporter genes. For the Ai2 vector, which lacks the WPRE, the CAG promoter was inserted between the first FRT and LoxP sites, and the cassette was cloned immediately downstream of the 5′ homology arm. The final targeting vectors contained 5′ and 3′ homology arms of 1.1 kb and 4.3 kb, as well as a PGK-DTA cassette for negative selection. Targeting constructs for knock-in Cre lines inserted into other gene loci were constructed in similar ways.
The targeting vectors were linearized and transfected into the 129/B6 F1 hybrid ES cell line G442 (link) using an Amaxa electroporator. G418-resistant ES clones were screened by Southern blot analysis of HindIII digested DNA, which was probed with a 1.1 kb genomic fragment from immediately upstream of the 5′ arm. We observed a recombination rate of about 25% for the four constructs. Positive ES clones were injected into C57BL/6J blastocysts to obtain chimeric mice following standard procedures. Both ES cell transfections and blastocyst injections were performed by the University of Washington Transgenic Resources Program. Due to the robustness of the G4 cells, high-percentage chimeras and high rates of germline transmission were routinely obtained. Chimeric mice were bred with either C57BL/6J mice to obtain germline transmission or various Cre-driver lines for direct characterization.
An Ai9 ES cell clone with strong germline transmission potency was used in subsequent transfections for the Flp-mediated exchange strategy outlined inSupplementary Figure 4 online. Ai9 ES cells were co-transfected using a Bio-Rad electroporator with 100 μg of pCAGGS-FLPe (Open Biosystems) and 40 μg of an incoming replacement vector. After 8 to 10 days of Hygromycin B selection, surviving colonies that also appeared green by fluorescence microscopy were picked and screened by PCR using primer sets designed to confirm a correct insertion of the incoming vector at the 5′ and 3′ FRT recombinase sites.
The targeting vectors were linearized and transfected into the 129/B6 F1 hybrid ES cell line G442 (link) using an Amaxa electroporator. G418-resistant ES clones were screened by Southern blot analysis of HindIII digested DNA, which was probed with a 1.1 kb genomic fragment from immediately upstream of the 5′ arm. We observed a recombination rate of about 25% for the four constructs. Positive ES clones were injected into C57BL/6J blastocysts to obtain chimeric mice following standard procedures. Both ES cell transfections and blastocyst injections were performed by the University of Washington Transgenic Resources Program. Due to the robustness of the G4 cells, high-percentage chimeras and high rates of germline transmission were routinely obtained. Chimeric mice were bred with either C57BL/6J mice to obtain germline transmission or various Cre-driver lines for direct characterization.
An Ai9 ES cell clone with strong germline transmission potency was used in subsequent transfections for the Flp-mediated exchange strategy outlined in