What are RCAs and how can I avoid them?
A. One concern when working with Adenoviral vectors is the possible occurrence of replication competent adenoviruses (RCAs) in a population of replication deficient adenoviruses (Ad). RCAs can emerge as a result of a double crossover event between the homologous overlapping sequences present in the recombinant Ad and the 293 genome (Lochmüller, 1994). This event results in the loss of the transgene and its replacement by the E1 region (Zhu, 1999) thus rendering the Ad replication competent without the need of a complementing cell line. Lochmüller et al. demonstrated that Ad stocks contained an increasing amount of RCAs after increasing number of passages in 293 cells. To avoid the emergence of E1-containing Ad, it is necessary to 1- always plaque purify the adenoviral stocks to be amplified, and 2- keep the passage number in 293 cells as low as possible, ideally no higher than 4. Included below is a list of references on occurrence, characterization and screening of RCAs in adenoviral
