Differential behavior of liposome-introduced specific RNAs in living Drosophila cells.
We have developed a protocol for efficiently introducing macromolecules into Drosophila tissue culture cells using liposomes. By carefully adjusting the fusion parameters, conditions have been established to routinely encapsulate 15-30% of the starting material into liposomes and to introduce 20-30% of the liposome-encapsulated material into the cells during a 30-minute fusion period. Essentially, all of the cells receive material from the liposomes and 10(9) cells can be fused at once. The fusion does not have any measureable effect on cell viability as assayed by trypan blue exclusion, growth rate, and cell morphology. We have utilized this technique to introduce radioactive RNAs into nonradioactive cells, thus enabling the behavior of the introduced RNAs to be followed unambiguously. Liposome-introduced small nuclear RNAs (snRNAs) are stable in the cell for at least 25 hours (approximately two cell generations), with 80% of the radioactivity remaining trichloroacetic acid (TCA) precipitable and the gel electrophoresis pattern remaining essentially unchanged. This is in contrast to liposome-introduced cytoplasmic RNAs, which are only 20% TCA precipitable after the first hour. In the cell, the introduced snRNAs attain a 10-35-fold higher concentration in the nucleus than the cytoplasm. Nuclear accumulation is not seen with Drosophila tRNA or 5S RNA, both of which attain the same nuclear as cytoplasmic RNA concentration.
Gross
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RH
Department of Biological Sciences, Dartmouth College, Hanover, NH 03755.
Rosen
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Cetron
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MS
D000818:Animals; D000936:Antigen-Antibody Complex / Q000302:isolation & purification; D002478:Cells, Cultured; D003593:Cytoplasm; D004331:Drosophila melanogaster / Q000235:genetics; D004337:Drug Carriers; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008081:Liposomes; D012313:RNA / Q000008:administration & dosage* / Q000302:isolation & purification
D000936:Antigen-Antibody Complex; D004337:Drug Carriers; D008081:Liposomes; D012313:RNA