Enhanced incorporation of transgenic DNA into zebrafish chromosomes by a retroviral integration protein.
Manufacture of lines of fish containing specific transgenes is difficult because most fish that hatch from embryos injected with foreign DNA are mosaic; few have the transgenic DNA integrated in germ-line cells. To determine whether the process of integration of exogenously supplied DNA into fish genomes could be accelerated, we examined the ability of the Moloney murine leukemia virus (MoMLV) integration protein (IN) to function in embryonic zebrafish cells. We used partially purified IN from a baculovirus/insect cell expression system and unpurified IN from extracts of psi-2 mouse cells that carry a MoMLV provirus. Both forms of IN were able to enhance expression in zebrafish 10 days after fertilization. At day 14 of development, fish injected with IN had higher levels of transgenic DNA than control fish. The ability of IN to enhance integration of transgenic constructs was demonstrated by a ligation-mediated polymerase chain reaction procedure, which was employed to detect junction fragments of foreign and host genomic DNA, generated by IN-mediated integration.
Ivics
Z
Z
Department of Genetics & Cell Biology, University of Minnesota, St. Paul 55108.
Izsvák
Z
Z
Hackett
P B
PB
D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000302:isolation & purification / Q000502:physiology*; D015151:Immunoblotting; D019426:Integrases; D008845:Microinjections; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012191:Retroviridae Proteins / Q000302:isolation & purification / Q000502:physiology*; D014162:Transfection / Q000379:methods*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*
D009841:Oligonucleotides; D012191:Retroviridae Proteins; D004247:DNA; D004254:DNA Nucleotidyltransferases; D019426:Integrases