AMPLIFIED AND TISSUE-DIRECTED EXPRESSION OF RETROVIRAL VECTORS USING PING-PONG TECHNIQUES

Ping-pong amplification is an efficient process by which helper-free r etrovirions replicate in cocultures of cell lines that package retrovi ruses into distinct host-range envelopes [11]. Transfection of a retro viral vector DNA into these cocultures results in massive virus produc tion, with potentially endless cross-infection between different types of packaging cells. Because the helper-free virus spreads efficiently throughout the coculture, it is unnecessary to use dominant selectabl e marker genes, and the retroviral vectors can be simplified and optim ized for expressing a single gene of interest. The most efficient ping -pong vector, pSFF, derived from the Friend erythroleukemia virus, has been used for high-level expression of several genes that could not b e expressed with commonly employed two-gene retroviral vectors. Contra ry to previous claims, problems of vector recombination are not inhere nt to ping-pong methods. Indeed, the pSFF vector has not formed replic ation-competent recombinants as shown by stringent assays. Here we rev iew these methods, characterize the ping-pong process using the human erythropoietin gene as a model, and describe a new vector (pSFY) desig ned for enhanced expression in T lymphocytes. Factors that limit tissu e-specific expression are reviewed.