Histone-mediated transfer and expression of the HIV-1 tat gene in Jurkat cells.
We studied the gene transfer efficiency of lipofection reagents in comparison to DEAE-Dextran. DOTAP, Dosper, and Lipofectin have lower transfection efficiency; Lipofectamine has a 2.5-fold better efficiency compared with DEAE-Dextran. We report a novel and highly efficient DNA transfer system based on the DNA-binding proteins histone 3 and histone 4. We have transferred the HIV-1 tat gene and measured the transactivation of HIV-1 LTR by the transactivator protein, expressed in Jurkat cells. The HIV-1 LTR was linked to the CAT gene as a reporter. Compared to DEAE-Dextran-mediated transfection, histone-mediated transfection resulted in a sevenfold higher expression of the CAT gene. The maximum transfection efficiency mediated by histones is dependent on the relative concentration (DNA:histone ratio) and the incubation time. In a gel-retardation assay, an optimal complex formation was observed under the same conditions that allowed the highest transfection efficiency. This ability of histones to increase the delivery and transgenic expression of foreign DNA in eukaryotic cells is not simply due to the positive ionic character of the histone proteins. Polylysine, histone H1, and histone H2A were unable to mediate gene transfection in our system. Monoclonal antibodies that recognize antigenic determinant present on all five histone proteins (anti-histone, pan) were able to neutralize the transfection-enhancing potential of histone 3 and histone 4. However, anti-histone IgG enhanced the retardation of mobility of histone-DNA complexes. The results of this study allow us to conclude that histones H3 and H4 can catalyze gene transfer and gene expression in eukaryotic cells without any requirement for additional constituents. For this reason, we have termed the new gene-delivery system as histonefection.
Demirhan
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I
Gustav-Embden Center of Biological Chemistry, Department of Molecular Biology, Frankfurt University Medical School, Germany.
Hasselmayer
O
O
Chandra
A
A
Ehemann
M
M
Chandra
P
P
D000818:Animals; D002411:Cation Exchange Resins / Q000378:metabolism; D015870:Gene Expression; D015696:Gene Products, tat / Q000096:biosynthesis / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000008:administration & dosage; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D050356:Lipid Metabolism; D008055:Lipids; D010714:Phosphatidylethanolamines / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D013997:Time Factors; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection / Q000379:methods*; D054322:tat Gene Products, Human Immunodeficiency Virus
D002411:Cation Exchange Resins; D015696:Gene Products, tat; D006657:Histones; D008055:Lipids; C086724:Lipofectamine; D010714:Phosphatidylethanolamines; D011994:Recombinant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; C020888:1,2-dielaidoylphosphatidylethanolamine