Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.

TLDR

The method is designed to streamline recombinant adenovirus production, enabling rapid development of improved Ad vectors. It inserts an expression cassette into the SwaI site of a full‑length adenoviral cosmid, cotransfects this cassette with a digested DNA‑terminal protein complex into 293 cells, and leverages the full‑length genome to allow easy genome modifications for E1‑substitution and E4‑insertion vector designs. The approach yields hundreds of clones per experiment, with roughly 70 % being the desired recombinant virus, and markedly reduces regeneration of the parental virus.

Abstract

An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with Eco T22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.