Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells.
Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST-Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST-Z2 is able to condense 130-150 kb bacterial artificial chromosomes (BACs) into protein-DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein-BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR(-) cells by GST-Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST-Z2-mediated gene transfer. Because DNA condensation by GST-Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.
Montigny
W J
WJ
Department of Pathology, University of Vermont, Burlington, VT 05405, USA.
Houchens
C R
CR
Illenye
S
S
Gilbert
J
J
Coonrod
E
E
Chang
Y C
YC
Heintz
N H
NH
D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D001692:Biological Transport; D016466:CHO Cells; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000737:chemistry; D016678:Genome; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics; D016335:Zinc Fingers
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C404766:REPIN1 protein, human; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA; D005982:Glutathione Transferase