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..../cell with a vaccinia virus recombinant expressing T7 RNA polymerase (vTF7-3, referred to in the text as vaccinia-T7) (32). Transfection mixes containing 0.5 ml of DMEM medium, 16 ml of transfectASE =-=(33)-=- and 3 mg of plasmid DNA were added to the cells 30 min after infection. For protein labelling, cells were starved for 30 min in DMEM minus methionine and cysteine (Gibco). This was then replaced with...
\"... Abstract: O- Alkyl derivatives of dioleoylphosphatidylcholine (DOPC) have been previously described as effective DNA transfection reagents. This communication reports the effects of the neutral helper lipid dioleoylphosphatidylethanolamine (DOPE) on the efficiency of transfection of BHK cells mediat ...\" Abstract - Add to MetaCart Abstract: O- Alkyl derivatives of dioleoylphosphatidylcholine (DOPC) have been previously described as effective DNA transfection reagents. This communication reports the effects of the neutral helper lipid dioleoylphosphatidylethanolamine (DOPE) on the efficiency of transfection of BHK cells mediated by the O-ethyl-, O-hexyl-, and O-octadecyl- DOPC derivatives, compounds that by themselves are known to exhibit lyotropic phase preferences of lamellar, lamellar or cubic (depending on conditions) and inverse hexagonal, respectively. The effect of DOPE on transfection efficiency was found to be inhibition of the ethyl compound, stimulation or inhibition (depending on amount of DOPE) of the hexyl compound and stimulation in the case of the octadecyl compound, i.e., DOPE had a beneficial effect on the lipids that formed non-lamellar phases. X-ray diffraction was used to determine the lyotropic phase of the lipid-DOPE mixtures and of the lipid-DNA complex. DNA-lipid complexes tended to be lamellar unless the lipids had a very strong tendency toward the hexagonal phase, in which case the DNA complex was also hexagonal. Thus, a mixture of equal amounts of DOPE and hexyl-DOPC formed a lamellar complex with DNA, although the lipids on their own assumed the hexagonal phase. Octadecyl-DOPC formed a hexagonal phase with DOPE and the 1:1 DOPE mixture formed a hexagonal phase DNA complex; however, if smaller amounts of DOPE were included, the complex had a lamellar structure, in contrast to the hexagonal phase of the lipids by themselves. For these cationic phospholipids, there was not necessarily a benefit to transfection of generating a hexagonal phase lipid-DNA complex. 52Citation Context
... less efficient than viral-based delivery systems, considerable effort has gone into synthesis of new lipids and examination of different formulation methods in the hope of improving their efficiency =-=[7,8]-=- We have developed a procedure to alkylate the phosphate oxygen of DOPC and so generate O-alkyl phospatidylcholinium compounds [9]. The ethyl ester of DOPC (EDOPC; 1,2-dioleoyl- sn-glycerol-3ethylphos...
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...ion, cells were ~60% confluent, well spread and uniformly distributed. Transfection was accomplished using a modification of the lipofection procedure described by Felgner et al. (44) and Rose et al. =-=(45)-=-. Lipofectin® was complexed with plasmid DNA according to the supplier’s suggestion with minor modifications as follows: plasmid DNA was diluted in EMEM and combined with an equal volume of diluted Li...
\"... We investigated the feasibility of transferring naked plasmid DNA containing a therapeutic gene (IL-12) into mice harboring growing Renca tumors. We found that naked DNA transferred into growing Renca and B16(F10) tumors gives higher expression level of reporter gene than complexes of DNA with DDAB/ ...\" Abstract - Add to MetaCart We investigated the feasibility of transferring naked plasmid DNA containing a therapeutic gene (IL-12) into mice harboring growing Renca tumors. We found that naked DNA transferred into growing Renca and B16(F10) tumors gives higher expression level of reporter gene than complexes of DNA with DDAB/ DOPE or DC-Chol/DOPE. Transfer of naked DNA carrying the IL-12 gene into growing Renca tumors causes a distinct therapeutic effect that depends on the time span between inoculation of mice with cancer cells and the beginning of the therapy. Therapy started on day 3 resulted in total cure (100%) of mice. Experimental gene therapy of cancer involves transfer of therapeutic DNA by means of various carriers. These can be divided into two broad categories: viral (adenoviruses and retroviruses) (for review see [1]), and nonviral (mostly cationic lipids) ones: [2]. In addition naked DNA can be inserted into target cells byCitation Context
... 1640 medium supplemented with 10% fetal bovine serum (Gibco BRL). Cultures were kept at 37�Cin5%CO 2 incubator. Cationic DNA carriers. As carriers of plasmid DNA we used cationic liposomes DDAB/DOPE =-=[19]-=- and DC-Chol/DOPE [20]. Liposomes were prepared at weight ratios of 0.6:1 (DDAB/DOPE) and 1:1 (DC-Chol/ DOPE). Liposome preparations (sonicated emulsions at 1 �g total lipids/�l H 2O) were kept at 4°C...
\"... 2. Applications of liposomes in basic sciences........................................ 493 ...\" Abstract - Add to MetaCart 2. Applications of liposomes in basic sciences........................................ 493Citation Context
...ds. First studies used cationic lipid dioleoyl-propyl-trimethylammonium (DOTMA) [57]. Later studies showed better transfection efficiencies by using some of the commercially available cationic lipids =-=[58]-=-. Better transfection efficiencies at reduced toxicity were found by using liposomes containing positively charged cholesterol [59]. Many novel cationic lipids are being synthetised in order to improv...
D Spadafora, D M Canter, R L Jackson, J Perrault, D. Spadafora, D. M. Canter, R. L. Jackson, J. Perrault \"... essential for transcription or replication. polymerase complex formation but is not stomatitis virus P protein modulates Constitutive phosphorylation of the vesicular ...\" Abstract - Add to MetaCart essential for transcription or replication. polymerase complex formation but is not stomatitis virus P protein modulates Constitutive phosphorylation of the vesicularCitation Context
...nstructs were carried out as before (21) using a cationic lipid mixture (dioleoyl-L-a-phosphatidylethanolamine and dimethyldioctadecylammonium bromide) prepared according to the method of Rose et al. =-=(28)-=-. The wild-type and mutant P constructs, as well as the wild-type L plasmid, were described previously (8, 21). The wild-type N gene construct was obtained by subcloning the XhoI fragment from the pSV...
E A Stillman, J K Rose, M A Whitt, Elizabeth A. Stillman, John K. Rose, Michael A. Whitt \"... structural proteins. stomatitis virus minigenomes encoding viral Replication and amplification of novel vesicular ...\" Abstract - Add to MetaCart structural proteins. stomatitis virus minigenomes encoding viral Replication and amplification of novel vesicularCitation Context
...ections were performed as described previously (38) with a suspension of liposomes composed of dimethyldioctadecyl ammonium bromide and L-a-dioleoylphosphatidylethanolamine at a weight ratio of 1:2.5 =-=(28)-=-. For cells grown in 35-mm-diameter dishes, we transfected 10 mg of plasmid encoding the VSV minigenome cDNAs with 5, 3, and 1 mg of plasmids encoding the N, P, and L proteins, respectively. Indirect ...
José M. Valpuesta, José L. Carrascosa, Juan Ortín, Amplificationof Nucleoprotein During VirusCitation Context
...). After 24 h of incubation at 37°C, the medium was replaced by 10 ml of DMEM containing 2% fetal bovine serum and incubated for further 24 h. Cationic liposomes were prepared as described previously =-=(65)-=-. RNP purification. Cultures infected and transfected as indicated above were collected 48 h after transfection and lysed for 2 h at 0°C in buffer A (10 mM Tris-HCl–1 mM EDTA–7.5 mM ammonium sulfate–0...
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...ation with purified His-hCLE protein (1/300 dilution). Immunofluorescence. Cultures of COS-1 cells were transfected with 5 mg of pHA-hCLE plasmid with a mixture of cationic liposomes (2 ml/mg of DNA) =-=(45)-=- in serum-free Dulbecco modified Eagle medium (DMEM). They were incubated for 6 h, washed with phosphate-buffered saline (PBS), refed with fresh DMEM containing 5% fetal calf serum, and used for analy...
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