AIM: 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. CONCLUSION: 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.
AIM: 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. CONCLUSION: 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: M J Robertson; R J Soiffer; S F Wolf; T J Manley; C Donahue; D Young; S H Herrmann; J Ritz Journal: J Exp Med Date: 1992-03-01 Impact factor: 14.307