Virus homologous recombination

Viral genome can be inserted with a large DNA sequence by homologous recombination, and the recombinant virus, which is defective in replication, can be plaque purified by using transcomplementing cells. However, this viral vector has several disadvantages it is difficult to obtain preparations that are completely defective in replication, the modified vector generates immune response and antibodies specific for HSV-1 are present. 

Kotin, R. M., Linden, R. M. and Berns, K. I. (1992). Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination. EMBO J. 11, 5071-5078. 

Because of the large size of the baculovirus genome, unique restriction sites are not available for simple replacement of the polyhedrin gene with the gene of interest. Thus, recombinant viruses are generated by homologous recombination. A number of different strategies are currently employed to this end. In the traditional protocol, Sf cells are... 

Merchilinsky M. Intramolecular homologous recombination in cells infected with temperature-sensitive mutants of vaccinia virus. J Virol. 1989 63 2030-2035. 

Alternative methods have been developed to obtain virus stocks without plaque purification for expression of recombinant proteins in infected insect cells. The Bac-to-Bac technology (Invitrogen, Thermo Fisher Scientific) avoids homologous recombination in insect cells by using site-specific transposition in E. coli. With this technology, recombinant bacmid-DNA is generated that is used to transfect insect cells. 

Fig. I. Schematic representation of homologous recombination with a replacement-type vector employing positive-negative selection. Exons are shaded and intronic regions are represented by open boxes. Vector sequences are denoted by a wavy line. HSV-tt, herpes simplex virus thymidine kinase expression vector P-Neo. promoter-neomyocin expression vector.

The presence of replication- competent virus in the vector preparation is problematic, since it could be associated with increased toxicity and may lacilitate the spread of the recombinant in vivo (18-20). Replication-competent adenovirus occurs by either cross contamination of the initial plaque with replication-competent vims from an adj acent plaque or reversion of the E1 deletion during the propagation of the recombinant by homologous recombination between sequences in the vector flanking the El deletion and the transfected El sequences in 293 cells. 

Isolating recombinant adenoviruses fiom molecular clones has many advantages. Virus that emerges fiom the transfection should only represent that derived from die molecular clone, thereby eliminating the need for screening of plaques, thereby saving a substantial amount of time. We have found that rescue of virus fiom a clone rarely fails in contrast to the method based on homologous recombination where we are unable to isolate a recombinant approximately 20% of the time. 

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