![]() ![]() This method is technically challenging because the Ad genome is large (around 36 kb) and contains few useful restriction sites that allow its assembly into a full-length molecular clone 8. ![]() The genome can then be manipulated in vitro and, following transfection into a packaging cell line, the virus can be rescued. The third method is based on direct cloning of the Ad genome into a plasmid vector. It is efficient but more complex and requires a three-step transformation, including the use of two different E. The commercially available pAdEasy system is based on this method. The second method is based on homologous recombination in Escherichia coli. However, recombination is relatively rare and the isolation of recombinant Ad vector using this method is laborious, time consuming and requires multiple rounds of plaque purification 7. Recombination between the shuttle vector and the Ad genome replaces the E1 domain with the gene of interest. In this method, the gene of interest is cloned into a shuttle vector and concomitantly transfected with the Ad genome into HEK 293 cells or other cells that provide E1 in trans. The most commonly used method is based on homologous recombination in a packaging cell line. Three major methods have traditionally been used to generate recombinant Ad vectors. Vectors based on rare human Ad serotypes and Ad from other species are being explored to overcome the impact of pre-existing immunity 1, 2, 3, 4, 5, 6. Pre-existing neutralizing antibodies dampen gene transfer efficacy and increase vector-mediated toxicity 2. This virus is endemic in most human populations, and neutralizing antibodies specific to AdHu5 can be detected in up to 40–60% of humans 1. Until now, most vectors are based on human serotype 5 (AdHu5). Vectors based on Ads are commonly used for gene transfer because of their broad tropism, high transduction efficiency and relatively low risk in safety. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |