Effective modifications for improved homologous recombination and high-efficiency generation of recombinant...

Generation of adenovirus-based vectors through homologous recombination within Escherichia coli cells is one of the most efficient strategies. A common challenge associated with this method is the formation of colonies containing self-ligated shuttle plasmid. To improve homologous recombination, a new pAdEasy-1-bearing competent cell line was constructed so that it no longer requires co-transformation with two plasmids and can generate more recombinant colonies (ninefold). New and efficient approaches were also tested to block shuttle plasmid self-ligation by a combined treatment of the plasmid with Taq DNA polymerase and calf intestine phosphatase (CIP) or blocking the formation of self-ligated plasmid-containing colonies by subcloning a suicide gene, ccdB, into the plasmid construct. Present experimental data show that these modifications are effective in eliminating self-ligated plasmid-containing colony background and offer greater simplicity, faster experimental progress, and higher efficiency in performing homologous recombination within E. coli cells, which could facilitate the production of high-titer infectious viral particles.