Magin-Lachmann C, Kotzamanis G, D'Aiuto L, Cooke H, Huxley C, Wagner E
Boehringer Ingelheim Austria GmbH, 1121 Vienna, Austria.
J Gene Med. 2004 Feb;6(2):195-209. doi: 10.1002/jgm.481.
The ability to deliver large (>100 kb) fragments of DNA to mammalian cells in vitro and in vivo is becoming increasingly important with the availability of BAC and PAC constructs for gene expression. Here we investigate in vitro and in vivo delivery of BACs up to 157 kb.
Different types of polyethylenimine (PEI) and Lipofectamine were used to deliver 150-kb BAC (bacterial artificial chromosome) DNA to mouse and human cell lines in tissue culture and the level of EGFP expression compared. To assess the intactness of the DNA delivered, a BAC carrying oriP/EBNA-1 was used to make stably transfected cell lines. Episomal DNA was then rescued into E. coli followed by analysis on a pulsed-field gel. Three different methods of in vivo delivery were also assessed for delivery of BAC DNA; intravenous injection of DNA/PEI particles, intramuscular injection with electroporation and high-volume injection into the tail vein.
PEI22 (linear polymer form, 22 kDa) was found to be the most efficient method for delivery of 150-kb BAC DNA to both cell lines in tissue culture. However, Lipofectamine 2000 was found to give a higher proportion of intact DNA than PEI22 in stably transformed colonies and almost all the DNA delivered by Lipofectamine 2000 was intact. Intravenous injection of DNA/PEI particles was found to be inefficient for delivery of BAC DNA. Intramuscular injection with electroporation of pure BAC DNA was very efficient and expression was maintained for 105 days. High-volume injection of BAC DNA gave excellent expression in the liver and intact BAC DNA could be rescued 7 days after injection.
These results demonstrate efficient delivery of intact, large (up to 157 kb) DNA constructs for in vitro gene expression and in vivo gene therapy applications.
随着用于基因表达的细菌人工染色体（BAC）和P1人工染色体（PAC）构建体的出现，在体外和体内将大的（>100 kb）DNA片段递送至哺乳动物细胞的能力变得越来越重要。在此，我们研究了长达157 kb的BAC在体外和体内的递送情况。
使用不同类型的聚乙烯亚胺（PEI）和脂质体转染试剂将150 kb的BAC（细菌人工染色体）DNA递送至组织培养中的小鼠和人类细胞系，并比较绿色荧光蛋白（EGFP）的表达水平。为了评估递送的DNA的完整性，使用携带oriP/EBNA-1的BAC构建稳定转染的细胞系。然后将游离型DNA拯救到大肠杆菌中，随后在脉冲场凝胶上进行分析。还评估了三种不同的体内递送方法用于BAC DNA的递送；静脉注射DNA/PEI颗粒、电穿孔肌肉注射和尾静脉大容量注射。
发现PEI22（线性聚合物形式，22 kDa）是在组织培养中将150 kb的BAC DNA递送至两种细胞系的最有效方法。然而，在稳定转化的菌落中，发现脂质体转染试剂2000比PEI22产生更高比例的完整DNA，并且脂质体转染试剂2000递送的几乎所有DNA都是完整的。发现静脉注射DNA/PEI颗粒对BAC DNA的递送效率低下。纯BAC DNA电穿孔肌肉注射非常有效，并且表达维持了105天。BAC DNA大容量注射在肝脏中产生了良好的表达，并且注射后7天可以拯救完整的BAC DNA。
这些结果证明了完整的大（高达157 kb）DNA构建体可有效递送至体外基因表达和体内基因治疗应用中。
