PANTROPIC RETROVIRAL VECTOR INTEGRATION, EXPRESSION, AND GERMLINE TRANSMISSION IN MEDAKA (ORYZIAS-LATIPES)

Pantropic retroviral vectors were used to introduce transgenes into Ja panese medaka (Oryzias latipes). These vectors contain the long termin al repeat (LTR) sequence of Moloney murine leukemia virus (Mo-MLV) and a reporter gene (neo or lacZ) regulated by the LTR sequence of rous s arcoma virus (RSV). Because these pseudotyped retroviral vectors conta in the vesicular stomatitis virus envelope glycoprotein (VSV-G), they have an extremely broad host cell range and can infect many no mammali an species. Newly fertilized medaka eggs (intact or dechorionated) wer e electroporated at different voltage settings in the presence of 4 x 10(4) cfu of pantropic retroviral vector. The survival rates of the pa ntropic retroviral vector-treated embryos ranged from 65% to 20% with increasing amplitude of electroporation. Dechorionation did not substa ntially affect the survival rate of embryos. PCR amplification demon s trated proviral sequences in up to 60% of the 2-month-old fish. The ef ficiency of gene transfer was enhanced by dechorionation. Furthermore, overnight incubation of dechorionated embryos with pantropic retrovir al vectors without electroporation also resulted in proviral integrati on in 60% of the embryos without compromising survival rate. Southern blot analysis of DNA samples isolated from polymerase chain reaction ( PCR) as positive F-reaction animals confirmed the integration of a sin gle copy of the provirus into the host genome. Three P-1 transgenic fe males transmitted the proviral sequence to 50% of their F-1 progeny in a back cross with wild-type males, suggesting that the entire germ:li ne of these P-1 fish was transformed by the pantropic retroviral vecto r. Expression of the neomycin phosphotransferase transgene in F-1 tran sgenic individuals was detected by reverse transcription (RT)-PCR ampl ification of the neo mRNA sequence. Furthermore, expression of a beta- galactosidase transgene was also observed in 4-day-old F-1 transgenic individuals. Thus, pantropic retroviral vectors provide a convenient m ethod to stably introduce and express foreign genes in medaka.