Comparison of Inserted Mouse Ip-10 Gene Copy Number in Helper-Dependent and Independent System Based on Piggybac Transposition in Human Embryonic Kidney Cells



Mirzapour H¹ ; Karamzade A¹ ; Khanahmad H¹ ; Salehi R¹ ; Kheirollahi M¹ Show Affiliations
Source: Journal of Isfahan Medical School Published:2014

Abstract

Background: A major bottleneck in the production of recombinant proteins in conventional linear method is the heterogeneity in number of transgene copies in the genome of the host cell that lead to variable levels of transgene expression. Aside from the low efficiency of the random integration, other phenomena such as positional effect contribute to low efficiency of transgene expression. PiggyBac is a class of DNA transposons which can transpose through cut and paste mechanism in host genome. Some specific characteristic of this transposon makes it promise vector in gene transfer studies. High capacity of transgene transposition and flexibility in molecular engineering of transposase are characteristics of PiggyBac transposons that are important in recombinant protein and gene therapy approaches. The aim of this study was estimation of transgene copy number based on helper-dependent and independent PiggyBac transposition system in human embryonic kidney (HEK) cells. Methods: Plasmid containing interferon gamma inducible protein 10 (IP-10) coding sequence flanked by PiggyBac terminal repeat element and plasmid containing transposase system and a unit plasmid containing both transposae and IP-10 coding sequence were used for generating recombinant cells in helper-dependent and independent system, respectively. Human embryonic kidney cells were transfected by each group. After clonal selection, absolute quantitative real-time polymerase chain reaction was used for estimation of transgene copy number. Findings: Estimation of IP-10 copy number has revealed 5 and 2 copies per cell in helper-independent and dependent system, respectively. Conclusion: Here, we report activity of PiggyBac transposition in human embryonic kidney cell line. PiggyBac helper-independent and dependent system was used for generation of stable cell line producing mouse IP-10 protein and our data confirmed permanent expression of this transgene with the mean of about 5 transgene copies per each cell.