Construction of Retro viral Packaging Cell Lines

Most of the time, retrovirus vectors retain only cis-acting sequences from the original viral genome. These sequences allow the recombinant structure to be transcribed (LTR promoter/enhancer) and the RNA to be processed (splicing and polyadenylation signals), packaged into a virion particle (packaging sequences), and replicated by the reverse transcriptase (tRNA binding site, R region, and polypurine track). The other viral functions have to be provided in trans for the assembly of recombinant viral particles to take place. This can be simply achieved by using a replication-competent helper virus, leading to the production of a mixed population. Nevertheless, helper-free stocks are desirable for most applications, since 1. The high frequency of recombination in a mixed virus stock is likely to lead to the appearance of recombinants with unknown structure and activity. These new chimeras, either spread by the helper virus or replication-competent themselves, create a potential safety problem. 2. Cell lineage analysis using retroviral marking can only be performed and interpreted in a helper-free context 3. In vivo gene transfer experiments can be jeopardized by disease (s) associated with helper-virus infection (1).