Efficient in-vitro transfer of a 60-Mb mammalian artificial chromosome into murine and hamster cells using cationic lipids and dendrimers.
Non-integrating artificial chromosomes represent a potentially promising approach to ex-vivo and in-vivo gene therapy applications. These large vectors require an efficient means for delivery to target cells. We have evaluated a panel of twenty-one commercially available transfection agents for their ability to mediate the in-vitro transfer of a 60-Mb murine artificial chromosome consisting of mouse major satellite DNA and a payload including a marker gene (hygromycin B) and a reporter gene (lacZ). A rapid screening procedure utilizing iododeoxyuridine-incorporated artificial chromosomes facilitated the assessment of different transfection conditions. The results were confirmed by cytogenetic analysis of positively transfected clones. By transfecting both hamster lung fibroblast cells (V79-4) and murine connective tissue cells [L-M(TK-)], the best results were obtained using either Superfect (cationic dendrimer) or LipofectAMINE 2000 (cationic lipid) with protocols adapted for metaphase chromosome preparation. Transfection efficiencies of 10(-4)-10(-2) (0.01-1%) were routinely observed, and recipient cells were able to maintain expression of the reporter gene over the total length of the experiment. This represents a significant advance over our previous attempts at mass-transfection of artificial chromosomes using microcell fusion, where we routinely achieved efficiencies at least two orders of magnitudes less than reported here. These data are particularly noteworthy given that lipid-mediated gene transfer typically involves transfecting millions of plasmids (1 microg of DNA from a 5 kb plasmid is approximately 1.2 x 10(11) copies) to each cell whereas the much larger artificial chromosomes comprise only a one-to-one ratio, yet achieve transfection efficiencies of (10(-2)-10(-1)), that is, comparable to our results. These data suggest that artificial chromosomes containing therapeutic genes can be successfully delivered to target cells in vitro using well-established transfection agents.
de Jong
G
G
Chromos Molecular Systems Inc., Burnaby, BC. gdejong@chromos.com
Telenius
A
A
Vanderbyl
S
S
Meitz
A
A
Drayer
J
J
D000818:Animals; D002460:Cell Line; D002470:Cell Survival; D022221:Chromosomes, Artificial, Mammalian / Q000235:genetics*; D003114:Colony-Forming Units Assay; D003239:Connective Tissue Cells / Q000166:cytology* / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007065:Idoxuridine / Q000378:metabolism; D066298:In Vitro Techniques; D008055:Lipids / Q000737:chemistry; D008168:Lung / Q000166:cytology; D051379:Mice; D014162:Transfection / Q000379:methods*
D008055:Lipids; D004247:DNA; D007065:Idoxuridine