title: Saturation of transgene protein synthesis from mRNA in cells producing a large number of transgene mRNA.
abstract: Experimental results have suggested that transgene expression can be saturated when large amounts of plasmid vectors are delivered into cells. To investigate this saturation kinetic behavior, cells were transfected with monitoring and competing plasmids using cationic liposomes. Even although an identical amount of a monitoring plasmid expressing firefly luciferase (FL) was used for transfection, transgene expression from the plasmid was greatly affected by the level of transgene expression from competing plasmids expressing renilla luciferase (RL). Similar results were obtained by exchanging the monitoring and competing plasmids. The competing plasmid-dependent reduction in transgene expression from the monitoring plasmid was also observed in mouse liver after hydrodynamic injection of plasmids. On the other hand, the mRNA and protein expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an endogenous gene, in the liver hardly changed even when transgene expression process is saturated. The expression of FL from a monitoring plasmid was significantly restored by siRNA-mediated degradation of RL mRNA that was expressed from a competing plasmid. These results suggest that the efficiency of protein synthesis from plasmid vectors is reduced when a large amount of mRNA is transcribed with no significant changes in endogenous gene expression.
authors.affiliation: Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan; telephone: +81-75-753-4580; fax: +81-75-753-4614.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Makiya
authors.initials: M
authors.lastname: Nishikawa
authors.forename: Naomi
authors.initials: N
authors.lastname: Takiguchi
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Suehara
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Takakura
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D018014:Gene Transfer Techniques; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic; D019076:Transgenes
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins