title: Modulation of the molecular conformation of a hepatocyte-targeting gene drug in order to improve its expression efficiency in vitro.
abstract: OBJECTIVE
  To construct different conformations of a plasmid DNA/vector complex (pcDNA3.1/IFN-gamma-ASOR-PLL) and transfect cells of the hepatoma cell line BEL7402 to investigate the optimal conformation of the complex for improved expression efficiency in the target cell.
  
  
  METHODS
  Double-distilled water and adjuvant were added to the naked pcDNA3.1/IFN-gamma, target vector ASOR-PLL and the ASOR-PLL-pcDNA3.1/IFN-gamma complex to create different conformations; molecules that were transfected into BEL7402 cells and the expression efficiency was determined by measuring the IFN-g concentration in the culture supernatant by ELISA.
  
  
  RESULTS
  Naked pcDNA3.1/IFN-gamma DNA distributed linearly in double-distilled water and condensed into a mica configuration in adjuvant; ASOR-PLL had a net-like distribution without adjuvant and a spider-like form in the adjuvant-treated group; the ASOR-PLL-pcDNA3.1/IFN-g complex had a divaricate form without adjuvant, but a bead-like or granular conformation in 0.1 and 0.2 mol/L of adjuvant, a homogeneous bacilliform or chromatoid-shaped conformation in 0.3 mol/L adjuvant, and varied shapes in 0.4 and 0.5 mol/L adjuvant. The supernatant IFN-gamma expression in the bacilliform/chromatoid conformation complex group was the highest among the different conformation groups and controls. When chloroquine was added the supernatant IFN-gamma concentration increased in the liposome group and decreased in the bacilliform/chromatoid conformation group .
  
  
  CONCLUSIONS
  The two structural molecules and their complex, ASOR-PLL-pcDNA3.1/IFN-gamma, were adjustable in the liquid mode. The specific bacilliform/chromatoid conformation of complex was lysosome enzyme-resistant and could play an active role in improving the efficiency of gene expression. The hypothesis that a chromosome-like conformation of the target gene molecule is involved in enhancing exogenous gene expression is proposed.
authors.affiliation: Department of Gastroenterology, the Second XiangYa Hospital, Central South University and Sheng Life Gene Drug Development Company, Changsha, Hunan Province, China.
authors.forename: Xiao Wei
authors.initials: XW
authors.lastname: Liu
authors.forename: Fang Gen
authors.initials: FG
authors.lastname: Lu
authors.forename: Xiao Ping
authors.initials: XP
authors.lastname: Wu
authors.forename: Chun Hui
authors.initials: CH
authors.lastname: Ouyang
authors.forename: Dong Ye
authors.initials: DY
authors.lastname: Yang
authors.forename: Yu
authors.initials: Y
authors.lastname: You
authors.forename: Guang Hui
authors.initials: GH
authors.lastname: Lian
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry* / Q000494:pharmacology*; D001212:Asialoglycoproteins / Q000737:chemistry* / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology*; D004247:DNA; D004337:Drug Carriers; D004797:Enzyme-Linked Immunosorbent Assay; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005822:Genetic Vectors; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry* / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology*; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology*; D010957:Plasmids; D011107:Polylysine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology*; D011487:Protein Conformation; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D001212:Asialoglycoproteins; D004337:Drug Carriers; D009961:Orosomucoid; C057326:asialoorosomucoid-polylysine conjugate; D011107:Polylysine; D007371:Interferon-gamma; D004247:DNA