Pantropic retroviral vector integration, expression, and germline transmission in medaka (Oryzias latipes).
Pantropic retroviral vectors were used to introduce transgenes into Japanese medaka (Oryzias latipes). These vectors contain the long terminal repeat (LTR) sequence of Moloney murine leukemia virus (Mo-MLV) and a reporter gene (neo or lacZ) regulated by the LTR sequence of rous sarcoma virus (RSV). Because these pseudotyped retroviral vectors contain the vesicular stomatitis virus envelope glycoprotein (VSV-G), they have an extremely broad host cell range and can infect many no mammalian species. Newly fertilized medaka eggs (intact or dechorionated) were electroporated at different voltage settings in the presence of 4 x 10(4) cfu of pantropic retroviral vector. The survival rates of the pantropic retroviral vector-treated embryos ranged from 65% to 20% with increasing amplitude of electroporation. Dechorionation did not substantially affect the survival rate of embryos. PCR amplification demonstrated proviral sequences in up to 60% of the 2-month-old fish. The efficiency of gene transfer was enhanced by dechorionation. Furthermore, overnight incubation of dechorionated embryos with pantropic retroviral vectors without electroporation also resulted in proviral integration in 60% of the embryos without compromising survival rate. Southern blot analysis of DNA samples isolated from polymerase chain reaction (PCR) as positive F1 reaction animals confirmed the integration of a single copy of the provirus into the host genome. Three P1 transgenic females transmitted the proviral sequence to 50% of their F1 progeny in a back cross with wild-type males, suggesting that the entire germline of these P1 fish was transformed by the pantropic retroviral vector. Expression of the neomycin phosphotranferase transgene in F1 transgenic individuals was detected by reverse transcription (RT)-PCR amplification of the neo mRNA sequence. Furthermore, expression of a beta-galactosidase transgene was also observed in 4-day-old F1 transgenic individuals. Thus, pantropic retroviral vectors provide a convenient method to stably introduce and express foreign genes in medaka.
Lu
J K
JK
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, USA.
Burns
J C
JC
Chen
T T
TT
D000818:Animals; D030801:Animals, Genetically Modified; D001358:Avian Sarcoma Viruses / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000502:physiology; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D008979:Moloney murine leukemia virus / Q000235:genetics; D009990:Oryzias / Q000235:genetics* / Q000821:virology*; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D012190:Retroviridae / Q000235:genetics*; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics; D016662:Virus Integration