Simplified Method for the Construction of Gene Targeting Vectors for Conditional Gene Inactivation in Mice
Peng Fei Wang, Dong Kong, Matthew W. Van Brocklin, Jun Peng, Chun Zhang, Stephanie J. Potter, Xiang Gao, Bin T. Teh, Nian Zhang, Bart O. Williams and Sheri L. Holmen
We have modified the MultiSite Gateway ® technology to generate mice with a condition-ally targeted allele of hrpt2 for the purpose of generating a mouse model of human hyper-parathyroidism- jaw tumor (HPT-JT) syn-drome. This technology allows for the simulta-neous cloning of large DNA fragments quickly and efficiently. The precise mechanism of site-specific recombination results in the assembly of multiple DNA segments in a defined order and orientation. Since the DNA fragments can be amplified directly from total genomic DNA, there is no need to isolate BAC clones or to subclone large segments of DNA by traditional techniques. This advantage, combined with the ability to assemble the components in parallel, greatly reduces the time required to construct the targeting vector, which is typically the rate limiting step in most gene-targeting projects. In this study, we demonstrate that the MultiSite Gateway ® technology can be used in combina-tion with both Cre/loxP- and Flp/FRT-mediated recombination to conditionally inactivate genes in mice.