In vitro and in vivo delivery of intact BAC DNA -- comparison of different methods.

Abstract

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.