The Adeno-X Expression System 1 uses a ligation-based method to generate adenoviral vectors for high-level protein expression in a wide variety of mammalian host cells. This Adeno-X expression system offers several major advantages over standard transfection and other adenoviral systems:
- High protein expression levels. Recombinant adenovirus containing your gene of interest infects target cells with multiple copies and high efficiency, cells transiently express the protein of interest at very high levels.
- Efficient infection of many mammalian cell types. Adenovirus efficiently infects a majority of human and many nonhuman cell types including mouse, rat, dog, chicken, rabbit, sheep, pig, and nonhuman primates (1–10). Adenovirus infects both dividing and nondividing cells.
- Optimized protocol for fast results. Instead of homologous recombination, this protocol uses an efficient, ligation-based method with pre-linearized, ready-to-ligate Adeno-X System 1 Viral DNA (see Images & Data tab). Our protocol has been optimized so that recombinant adenovirus can be generated faster than with other methods (see Images & Data tab). The system also provides a convenient shuttle vector, pShuttle2, and a unique double digestion buffer that saves time during cloning.
- Simple cloning procedures. With our optimized ligation-based approach, the Adeno-X System 1 eliminates nonrecombinant adenovirus and produces recombinant adenovirus much more rapidly and reliably than conventional homologous recombination approaches (see Table I in the Images & Data tab), without having to switch bacterial strains.
To produce recombinant adenovirus, insert your gene of interest into the multiple cloning site of the pShuttle2 vector’s expression cassette. Then, transfer the cassette into the ligation-ready Adeno-X Viral DNA using the two extremely rare restriction enzyme sites, PI-SceI and I-CeuI. Subsequent digestion with SwaI removes self-ligated or nonrecombinant adenoviral DNA. Transform E. coli strain with the ligation mixture and identify recombinant clones by restriction enzyme digests. Then transfect a low-passage HEK 293 cell line with linearized recombinant adenoviral DNA and harvest recombinant adenovirus several days later.