title: Study on relationship between expression level and molecular conformations of gene drugs targeting to hepatoma cells in vitro.
abstract: OBJECTIVE
  To increase exogenous gene expression level by modulating molecular conformations of targeting gene drugs.
  
  
  METHODS
  The full length cDNAs of both P(40) and P(35) subunits of human interleukin 12 were amplified through polymerase chain reaction (PCR) and cloned into eukaryotic expressing vectors pcDNA3.1(+/-) to construct plasmids of P(+)/IL-12, P(+)/P(40) and P(-)/P(35). These plasmids were combined with ASOR-PLL to form two targeting gene drugs [ASOR-PLL-P(+)/IL-12 and ASOR-PLL-P(+)/P(40) + ASOR-PLL-P(-)/P(35)] in optimal ratios. The conformations of these two drugs at various concentrations adjuvant were examined under electron microscope (EM) and the drugs were transfected into HepG2 (ASGr+) cells. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed with total RNA extracted from the transfected cells to determine the hIL12 mRNA transcript level. The hIL12 protein in the cultured supernatant was measured with enzyme-linked immunosorbent assay (ELISA) 48 hours after transfection.
  
  
  RESULTS
  Targeting gene drugs, whose structures were granular and circle-like and diameters ranged from 25 nm to 150 nm, had the highest hIL-12 expression level. The hIL-12 expression level in the group co-transfected with ASOR-PLL-P(+)/P(40) and ASOR-PLL-P(-)/P(35) was higher than that of ASOR-PLL-P(+)/IL-12 transfected group.
  
  
  CONCLUSIONS
  The molecular conformations of targeting gene drugs play an important role in exogenous gene expression level, the best structures are granular and circle-like and their diameters range from 25 nm to 150 nm. The sizes and linking styles of exogenous genes also have some effects on their expression level.
authors.affiliation: Department of Gastroenterology, Xiangya Second Hospital, Central South University, Changsha 410011, Hunan, China.
authors.forename: Dong-Ye
authors.initials: DY
authors.lastname: Yang
authors.forename: Fang-Gen
authors.initials: FG
authors.lastname: Lu
authors.forename: Xi-Xiang
authors.initials: XX
authors.lastname: Tang
authors.forename: Shui-Ping
authors.initials: SP
authors.lastname: Zhao
authors.forename: Chun-Hui
authors.initials: CH
authors.lastname: Ouyang
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Wu
authors.forename: Xiao-Wei
authors.initials: XW
authors.lastname: Liu
authors.forename: Xiao-Ying
authors.initials: XY
authors.lastname: Wu
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000628:therapy*; D018390:Gene Targeting; D015316:Genetic Therapy; D006801:Humans; D018664:Interleukin-12 / Q000737:chemistry* / Q000235:genetics*; D008113:Liver Neoplasms / Q000628:therapy*; D008968:Molecular Conformation
chemical_list: D018664:Interleukin-12