Lee J T, Watarai S, Kakidani H, Onuma M, Zhao D D, Yasuda T
Department of Cell Chemistry, Okayama University Medical School, Japan.
J Vet Med Sci. 1997 Mar;59(3):169-74. doi: 10.1292/jvms.59.169.
We investigated whether cationic liposomes are efficient at delivering the gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (pLTR-DT) into BLV-infected cells and are also suitable for in vivo use. The transfection activity of the cationic liposomes composed of N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dioleoyl phosphatidylethanolamine (DOPE) and dilauroyl phosphatidylcholine (DLPC) (1:2:2, molar ratio) (TMAG-liposome) and liposomes composed of phosphatidylserine (PS) (PS-liposome) was evaluated by the luciferase assay using a plasmid which contains the coding sequence of firefly luciferase under the control of the SR alpha promoter (pSR alpha/L-A delta 5). The TMAG-liposome gave highly efficient transfection in the presence of serum. On the other hand, PS-liposome showed inferior efficiency. When BLV-infected cells were co-transfected with a fixed amount of pSR alpha/L-A delta 5-entrapped TMAG-liposome and various amount of pLTR-DT-containing TMAG-liposome, the luciferase activity in the BLV-infected cells was inhibited by the addition of pLTR-DT-entrapped TMAG-liposome dose-dependently. The cationic TMAG-liposome containing pLTR-DT was successively added to BLV-infected cells in culture. The number of viable cells was markedly reduced by the cationic TMAG-liposome containing pLTR-DT. On the other hand, TMAG-liposome containing pSR alpha/L-A delta 5 showed no such effect. pLTR-DT entrapped by the cationic TMAG-liposome was not digested by the treatment with DNase I and with serum. These results suggest that the cationic liposomes, such as TMAG-liposome, may be efficient transfection reagent for the BLV-infected cells and can be utilized for DT-A gene delivery into the BLV-infected cells in vivo.
我们研究了阳离子脂质体在将牛白血病病毒（BLV）长末端重复序列（LTR）控制下的白喉毒素A链（DT-A）基因（pLTR-DT）递送至BLV感染细胞中时是否高效，以及是否适用于体内应用。通过使用含有萤火虫荧光素酶编码序列且受SRα启动子控制的质粒（pSRα/L-Aδ5）进行荧光素酶测定，评估了由N-（α-三甲基铵乙酰基）-二癸基-D-谷氨酸氯（TMAG）、二油酰磷脂酰乙醇胺（DOPE）和二月桂酰磷脂酰胆碱（DLPC）（摩尔比1:2:2）组成的阳离子脂质体（TMAG-脂质体）以及由磷脂酰丝氨酸（PS）组成的脂质体（PS-脂质体）的转染活性。TMAG-脂质体在有血清存在的情况下能实现高效转染。另一方面，PS-脂质体的效率较低。当用固定量的包裹有pSRα/L-Aδ5的TMAG-脂质体和不同量的含有pLTR-DT的TMAG-脂质体共转染BLV感染细胞时，BLV感染细胞中的荧光素酶活性会因加入包裹有pLTR-DT的TMAG-脂质体而呈剂量依赖性受到抑制。将含有pLTR-DT的阳离子TMAG-脂质体连续添加到培养的BLV感染细胞中。含有pLTR-DT的阳离子TMAG-脂质体使活细胞数量显著减少。另一方面，含有pSRα/L-Aδ5的TMAG-脂质体则无此效果。阳离子TMAG-脂质体包裹的pLTR-DT经DNA酶I和血清处理后未被消化。这些结果表明，诸如TMAG-脂质体之类的阳离子脂质体可能是用于BLV感染细胞的高效转染试剂，并且可用于在体内将DT-A基因递送至BLV感染细胞。
