The transfected cells must also be over infected with a helper virus, e.g., Adenovirus, which can contaminate the rAAV stocks. To overcome this problem, a packaging/helper plasmid containing all AAV and Adenovirus functions required for amplification and packaging of AAV vector constructs has been generated. Even if the production of helper virus-free rAAV stocks is obtainable, the titer of viral stocks is still highly dependent on transfection efficiency and on the particular human cell line utilized. An alternative method has been developed using the Baculovirus to provide the functions necessary for rAAV. In view of the greater complexity of the cell biology and genetics of metazoans, we explored the possibility to obtain AAV genome replication in the yeast Saccharomyces cerevisiae. Thanks to the high evolutionary conservation of fundamental biochemical pathways, yeast has been and is currently used to clarify biological processes of multicellular eukaryotic organisms. Yeast offers the advantage to be easily cultured and genetically manipulated. Moreover, this microorganism has already demonstrated its usefulness for virus research: many RNA or DNA viruses GSK1120212 infecting plants , animals or humans , replicate in yeast. Furthermore, yeast has been utilized to produce Reversine vaccines for Hepatitis B and for Papilloma viruses , for drug discovery and to elucidate the function of individual proteins from important pathogenic viruses such as HIV, Hepatitis C virus and Epstein2Barr virus. The AAV genome inserted into a plasmid vector can initiate a productive AAV replication when it is transfected in human cells that are simultaneously or subsequently infected with a helper virus. The AAV genome is released from a circular plasmid in a way that is similar to the rescue of the integrated AAV provirus in latent phase. It has also been observed that the rescue of the AAV genome in HeLa cells extracts is more efficient when the Rep68 protein is expressed. We, therefore, checked if Rep proteins expressed from pAAVRepURA were sufficient to rescue AAV genome from the circular plasmid in yeast. To do so, low Mr DNA from URA3+yeast clones transformed with the pAAVRepURA was analyzed by Southern blot and probed with URA3 gene to check for the presence of rescued ssDNA that is expected to be about 3 kb. This analysis revealed the presence of only a band of,6 kb in one clone and a band with a molecular weight higher than 10 kb in the another clone.