Amplified and tissue-directed expression of retroviral vectors using ping-pong techniques.
Ping-pong amplification is an efficient process by which helper-free retrovirions replicate in cocultures of cell lines that package retroviruses into distinct host-range envelopes [11]. Transfection of a retroviral vector DNA into these cocultures results in massive virus production, 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 selectable marker genes, and the retroviral vectors can be simplified and optimized 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 be expressed with commonly employed two-gene retroviral vectors. Contrary to previous claims, problems of vector recombination are not inherent to ping-pong methods. Indeed, the pSFF vector has not formed replication-competent recombinants as shown by stringent assays. Here we review these methods, characterize the ping-pong process using the human erythropoietin gene as a model, and describe a new vector (pSFY) designed for enhanced expression in T lymphocytes. Factors that limit tissue-specific expression are reviewed.
Hoatlin
M E
ME
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201-3098, USA.
Kozak
S L
SL
Spiro
C
C
Kabat
D
D
D000818:Animals; D004921:Erythropoietin / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics; D011533:Proviruses / Q000235:genetics / Q000502:physiology; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012190:Retroviridae / Q000235:genetics* / Q000502:physiology; D013155:Spleen Focus-Forming Viruses / Q000235:genetics / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured
D004921:Erythropoietin