Journal of Isfahan Medical School

Journal of Isfahan Medical School

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

Document Type : Original Article (s)

Authors
Hadi Mirzapour 1
Arezo Karamzade 1
Hossein Khanahmad 2
Rasoul Salehi 3
Majid Kheirollahi 4
1 MSc Student, Department of Genetics and Molecular Biology, School of Medicine AND Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Iran
2 Assistant Professor, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
3 Associate Professor, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
4 Associate Professor, Pediatric Inherited Diseases Research Center AND Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
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.
Keywords
Piggybac transposon
Gene therapy
Recombinant protein
Transgene copy number
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Journal of Isfahan Medical School
Volume 32, Issue 275 - Serial Number 275
January and February 2014
Pages 170-181

  • Receive Date 28 October 2013
  • Accept Date 06 April 2022