Gene therapy using non-viral gene delivery vehicle might be applied for the treatment of viral infection, cancer and genetic disorders. However, the most challenging barrier is the low efficiency of transgene expression. Therefore, optimization of transgene expression is very important and critical to be performed before doing extensive evaluation of the transfection agent. Herein, we present transgene expression optimization data of gene encoding luciferase condensed with a series of short fully synthesized lipopeptide on COS7, HeLa and 293T cells. The complex formation, effect of molar ratio, forced sedimentation, incubation period of the DNA-transfection agent complexes were explored on transfection efficiency. Moreover, the enhancement effect in transfection efficiency of polymer PEI as co-transfection agent in DNA-lipopeptide was also investigated. The results show that the complex particles of the DNA-lipopeptide were formed efficiently in the low ionic strength environment (HEPES Glucose Buffer pH 7.4), and the complex was incubated more than 10 hours before it was transfected onto the cells. Depending on the lipopeptide, the DNA-lipopeptide incubation in 24 hours (at 4-6oC) resulted an increased up to 2-4-fold in the transgene expression. In addition, polymer PEI enhanced the transfection efficiency of the lipopeptide-mediated gene delivery up to 50-fold compared to control. In summary, short and a linear of fully synthesized lipopeptide-based transfection agent facilitated transgene expression in several mammalian cells by optimizing the DNA-lipopeptide complex formation and transfection condition
