A Novel Method for Transfection and Expression of Reconstituted DNA-Protein Complexes in Eukaryotic Cells

Abstract

Transfection of foreign DNA into eukaryotic cells has become an important tool in molecular biology. Based on the results of previous studies of the core structure of human adenoviruses, we have developed a novel transfection method. The procedure involves the in vitro reconstitution of foreign DNA-of viral or other origins-with the major core protein VII of adenovirus type 2 (Ad2) or protamine from salmon sperm. Both proteins are rich in basic amino acids and appear to share structural features. The DNA-protein complexes are added directly to the medium of eukaryotic cells. The in vitro formation of specific DNA-protein complexes can be assessed by gel electrophoretic analyses. Bovine serum albumin does not enter into specific complexes with DNA. Transfection of DNA-protein VII or DNA-protamine complexes results in their rapid transport into the cell nuclei. About 2-4 hr after transfection, up to 40% of the DNA added to cell cultures in complexes can be found in the nucleus, as compared with less than 10% of the DNA when other transfection methods are applied or when naked DNA is added to cell cultures. DNAs transfected by the new method into mammalian or insect cells retain their characteristic restriction patterns at least 48 hr after transfection and are expressed efficiently. Supercoiled circular plasmid DNAs are converted to open circular or linear DNA. Expression has been measured both for transiently expressed genes (chloramphenicol acetyltransferase gene, Ad2 DNA in human HeLa cells) and for genes that have been integrated into the host genome and are expressed permanently, such as the gene for neomycin phosphotransferase in hamster BHK21 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular trafficking of nonviral vectors

“…221 Similar to whole Ad, gene transfer was inhibited by WGA, suggesting that interaction with the NPC, likely via the hexon, enables gene transfer. The core protein VII of Ad2, which normally binds viral genomic DNA and facilitates its transport into the nucleus, 222 enabled nuclear association of about 40% of applied DNA by 2-4 h after transfection, as distinguished by subcellular fractionation, 222 though it was unclear whether the DNA was actually transported into the nucleus. Vpr of HIV has both DNA condensing and nuclear targeting features that can facilitate nonviral gene transfer.…”

Improvement of exogenous DNA nuclear importation by nuclear localization signal‐bearing vectors: a promising way for non‐viral gene therapy?

“…Other cationic peptides with a less canonical NLS sequence (with regard to that of SV40 antigen T), such as protamine (Wienhues et al, 1987), histone H2 (Balicki et al, 2002) and melittin-derived peptides , have been shown to display DNA compaction and enhanced in vitro transfection efficiency properties.…”

An Imaging System to Monitor Efficacy of Adenovirus-Based Virotherapy Agents

“…Whether the increased thermostability pertains to the entire adenovirus structure or just the core is uncertain. It is possible that a more thermostable core alone would produce the results we obtained since a DNA-pVII complex by itself has transfection capability (Wienhues et al, 1987). Such "nonviral" transduction would likely contribute to the increased t.u.…”