RESUMO
Histone H2A can deliver transgenic DNA into mammalian cells in vitro. The ability of DNA delivery in vivo is a question of the further work, but it is possible to estimate factors in in vitro experiments, which affect delivery in vivo. The first step in this direction was to determine sizes and ζ-potentials of histone H2A complexes with DNA. In this work, we produced recombinant histone H2A and its modification, containing TAT-peptide from human immunodeficiency virus TAT protein, which is capable of enhancing macromolecule delivery into mammalian cells. The effective diameters and ζ-potentials of histones-DNA complexes were estimated. Complexes of histone H2A and complexes of modified histone H2A-TAT with DNA had positive ζ-potentials. Complexes of histone H2A with DNA had an effective diameter of about 200 nm. Histone modification with TAT-peptide led to aggregation and formation of massive particles of about 1 µm in diameter.
Assuntos
DNA/química , Histonas/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , HIV-1/química , HIV-1/genética , Histonas/genética , Humanos , Tamanho da Partícula , Plasmídeos/química , Plasmídeos/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Simultaneous expression of multiple target genes is often required in biotechnology. Multicistronic vectors coding for several proteins are being actively developed for this purpose. In commercially available vectors different variants ofencephalomyocarditis virus internal ribosome entry site (IRES EMCV) are used most often. However, many researchers consider that utilization ofself-cleaving 2A peptides sequences within multi- and bicistronic vectors is more promising. In this work, we compared the efficiency of gene expression in cells transfected with bicistronic vectors based on IRES EMCV and 2A peptide sequence derived form porcine teschovirus-1 (P2A). Efficiency ofgene expression was determined in three mammalian cell lines by measurement of co-expression levels of genes coding for RFP and EGFP proteins linked by IRES or P2A sequence. Higher level oftransgene expression was exhibited by cells transfected with the vector containing the 2A peptide sequence.
