Ligation of exogenous linear DNA after gene transfer in vitro and in vivo

✍ Scribed by Karola Rittner; Annie Benavente; Eric Jacobs

• Publisher: John Wiley and Sons
• Year: 2003
• Tongue: English
• Weight: 121 KB
• Volume: 5
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.406

✦ Synopsis

Abstract

Background

We have analyzed the physical/topographical state of linear exogenous DNA after gene transfer in vitro and in vivo.

Methods and results

Linear DNA carrying a luciferase expression cassette, either intact or corrupted within the coding region, was tested in gene transfer experiments in vitro and in vivo. To this, a plasmid with a CMV‐IE1 promoter‐driven luciferase gene was rendered non‐functional by the insertion of a 1.2 kb __Eco__RV–__Eco__RV fragment. After removal of the insert by digestion with __Eco__RV, the resulting linear DNA fragments were used to transfect HeLa cells. The recovery of luciferase activity from these cells indicated functional reconstitution of the expression cassette. Recovery of low molecular weight DNA from HeLa cells allowed amplification of an intact luciferase gene, confirming accurate ligation of free DNA ends. In the mouse, rapid intravenous injection of plasmid DNA, linearized within the luciferase gene, resulted in significant luciferase activities in liver and lung. Ligation products could be detected by PCR.

Conclusions

These data suggest that linear DNA is efficiently circularized after gene transfer in vitro and in vivo. Secondly, equally high luciferase activities were observed in the mouse after rapid intravenous injection of luciferase expression cassettes, either consisting of linear DNA produced by PCR, or carried by linearized plasmid DNA. These findings encourage the use of linear DNA elements for gene transfer applications in vivo. Copyright © 2003 John Wiley & Sons, Ltd.