Abstract
We established a simple and efficient method for gene transfer in vitro (to cultured cells) and in vivo (to an adult organ) using liposomes. Plasmid DNA and proteins were efficiently co-encapsulated in liposomes by agitation and sonication, and were co-introduced into cells by hemagglutinating virus of Japan (HVJ)-mediated membrane fusion. Introduction of the Escherichia coli beta-galactosidase gene with non-histone chromosomal protein high mobility group 1 (HMG1) into LLCMK2 cells resulted in about 3 times higher beta-galactosidase activity than that on introduction of the gene alone. Two days after injection of HVJ-liposomes containing the beta-galactosidase gene and HMG1 under the perisplanchnic membrane of adult rat liver, hepatic cells near the injection site were found by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to have beta-galactosidase activity. After similar injection of HVJ-liposomes containing the hepatitis B virus surface antigen (HBsAg) gene and HMG1, HBsAg was detected in the serum for 9 days with a maximum of 25-45 ng/ml on day 2 after the injection.
Highlights
Introduction of DNAHMGl Compkx into Cultured Cells25.2 or Adult Rat Liver 16,000 100:3216,000 100:320 2.016,000 10032' 0 100:32After incubation of 1 X lo[6] LLCMK2 cells with 2 ml of purifiedLLCMKZ cells were maintained in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal calf serum. 1 X lo6cells were inoculated into 100-mmPetri dishes on day 0
We previously reported that non-histone nuclear protein (HMG1) facilitates migration of plasmid DNA into the nuclei when co-introduced with the plasmid DNA into thecytoplasm by hemagglutinating virus of Japan (HVJ)-liposome-red blood cell ghosts ( l l ), and in this way we succeeded in expressing a foreign gene efficiently in liver cells of adult rats (11, 12)
Liposomes (5 mgof lipids and 5-20pgof encapsulated DNA) was HVJ-liposome (5 mgof lipids), the 0-galactosidase activity in cell added to the dishes
Summary
Keiko Kato, MahitoNakanishiS, Yasufumi Kaneda, Tsuyoshi Uchidat, and Yoshio Okada. From the Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565, Japan. We previously reported that non-histone nuclear protein (HMG1) facilitates migration of plasmid DNA into the nuclei when co-introduced with the plasmid DNA into thecytoplasm by HVJ-liposome-red blood cell ghosts ( l l ) , and in this way we succeeded in expressing a foreign gene efficiently in liver cells of adult rats (11, 12). Construction of Plasmids pActlacZ-pAct-c-myb (a gift from Dr Ishii, the Instituteof Physical and Chemical Research), which contained the 5’-promoter region (370 base pairs) and the first intron (900 base pairs) of the chicken 0-actin gene (13), was restricted with XhoIIBamHI and cloned into the SaEIIBamHI site of pUC19. The XhoIIEcoRI fragment containing HBsAg (ORF S, nucleotides 1-1856) of pSV2-LMSwas inserted into the XhoIIEcoRI site of this plasmid
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