Large macromolecules can be introduced into cultured mammalian cells using erythrocyte membrane vesicles.
Plasmid 6.4 kbp DNA, 14 kbp DNA, lambda phage particles, all of which contained herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene, or IgM molecules, were mixed with erythrocyte membranes and treated with neutral detergent. The transparent mixture was diluted with phosphate-buffered saline (PBS), followed by centrifugation to collect membrane vesicles containing the large macromolecules. 10-15% of 6.4 kbp, 3% of 14 kbp, 4-7% of the lambda phage particles and 14.5% of IgM were trapped within erythrocyte membrane vesicles. The membrane vesicles containing these molecules were fused with L cells, or rat F2408#20 cells, both of which are deficient in thymidine kinase activity. In each case, transformants were obtained. 2 X 10(5) - 7 X 10(5) phage PFU or 1.5 X 10(6) - 8 X 10(7) DNA molecules were required to obtain one transformant from L cells, but 2-3 X 10(7) phage PFU or 2 X 10(9) - 1 X 10(10) DNA molecules were required for one transformant from rat cells. Number of colonies which transiently expressed TK genes in L cells was also determined by autoradiography. The ratio of stable transformants to colonies positive for transient expression in cells treated with low doses of DNA or lambda phage was 46-68%. The transformation efficiency of human fibroblast cells by pSV2-gpt DNA trapped in erythrocyte membrane vesicles was less than that of L cells by HSV-TK DNA, but almost the same as that of rat cells by HSV-TK DNA.
Sugawa
H
H
Uchida
T
T
Yoneda
Y
Y
Ishiura
M
M
Okada
Y
Y
D000818:Animals; D010582:Bacteriophage lambda; D002478:Cells, Cultured; D003584:Cytological Techniques; D004910:Erythrocyte Membrane; D006801:Humans; D007075:Immunoglobulin M; D007739:L Cells; D008561:Membrane Fusion; D051379:Mice; D010957:Plasmids; D051381:Rats; D013937:Thymidine Kinase / Q000235:genetics
D007075:Immunoglobulin M; D013937:Thymidine Kinase