P-transposable vectors expressing a constitutive and thermoinducible hsp82-neo fusion gene for Drosophila germline transformation and tissue-culture transfection.
Three P-transposable vectors (approx. 16, 12, and 9 kb) were constructed containing a hsp82-neo fusion gene encoding a truncated heat-shock protein 82 of Drosophila pseudoobscura and the bacterial neomycin phosphotransferase (NPT). In transgenic Drosophila melanogaster, hsp82-neo exhibits high levels of housekeeping gene promoter and NPT activities in all cells in the absence of heat-shock and is further induced (fivefold) by elevated temperatures (35 degrees-36 degrees C). The hsp82-neo selection of transformants is possible from embryo to adulthood. The hsp82-neo insertion in a P-element plasmid carrying an alcohol-dehydrogenase-encoding gene (Adh) produced plasmids pHS22 (approx. 16 kb) and pHS24 (approx. 12 kb), in which both genes were expressed, as observed in 13 transgenic strains. Cloning of DNA fragments up to at least 16 kb in a third vector, pHS85 (approx. 9 kb), lacking the Adh cointegrate is facilitated by a 104-bp multiple cloning site (MCS) positioned downstream (3') from hsp82-neo. To accept inserts of nonselectable foreign genes, MCS provides 20 restriction sites, eight of them unique. The hsp82-neo-expressing vectors also function in cell-culture transfection assays. The hsp82-neo fusion gene (3.73 kb) may be of wide application as a dominant selection marker in other animal systems and plants.
Sass
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Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138.
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D004251:DNA Transposable Elements; D004331:Drosophila melanogaster / Q000235:genetics*; D005091:Exons; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000235:genetics*; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000235:genetics*; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014162:Transfection; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D006360:Heat-Shock Proteins; D000426:Alcohol Dehydrogenase; D010770:Phosphotransferases; D019868:Kanamycin Kinase