Red cell ghost-mediated microinjection of RNA into HeLa cells. I. A comparison of two techniques for the entrapment and microinjection of tRNA and mRNA.
We have compared two techniques for introducing RNA into red blood cell ghosts. In the pre-swell technique, RNA is introduced into red cells without prior removal of endogenous contents. In the multiple lysis technique, the red cells are subjected to two or three cycles of of reversible lysis, prior to introducing the RNA, in order to first remove the normal red cell constituents. The pre-swell technique offers much greater entrapment of both tRNA and protamine messenger RNA (mRNA), but the RNA appears to be degraded during the procedure. This may be due either to nucleolytic degradation or oxidation by the high concentration of endogenous hemoglobin. The multiple lysis technique offers much lower entrapment but also results in diminished degradation of the entrapped RNA. Although some degradation is apparent, a significant portion of the biological activity of the entrapped protamine mRNA is retained. We have also fused red cells loaded with protamine mRNA by the multiple lysis technique to HeLa cells using polyethylene glycol 6000. The recipient HeLa cells are capable of translating this heterologous message into protamine, a trout testis chromosomal protein.
D002459:Cell Fusion; D004910:Erythrocyte Membrane; D004912:Erythrocytes; D006367:HeLa Cells; D006461:Hemolysis; D006801:Humans; D008845:Microinjections / Q000379:methods*; D011092:Polyethylene Glycols; D014176:Protein Biosynthesis; D012313:RNA / Q000008:administration & dosage*; D012333:RNA, Messenger / Q000008:administration & dosage; D012343:RNA, Transfer / Q000008:administration & dosage
D012333:RNA, Messenger; D011092:Polyethylene Glycols; D012313:RNA; D012343:RNA, Transfer