RESUMO
Objective: Genetic modification of human embryonic stem cells [hESCs] is critical for their extensive use as a fundamental tool for cell therapy and basic research. Despite the fact that various methods such as lipofection and electroporation have been applied to transfer the gene of interest [GOI] into the target cell line, however, there are few reports that compare all parameters, which influence transfection efficiency. In this study, we examine all parameters that affect the efficiency of electroporation and lipofection for transient and long-term gene expression in three different cell lines to introduce the best method and determinant factor
Materials and Methods: In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for transient expression of green fluorescent protein in two genetically different hESC lines, Royan H5 [XX] and Royan H6 [XY], as well as human foreskin fibroblasts [hFF]. For long-term EGFP expression VASA and OLIG2 promoters [germ cell and motoneuron specific genes, respectively], were isolated and subsequently cloned into a pBluMAR5 plasmid backbone to drive EGFP expression. Flow cytometry analysis was performed two days after transfection to determine transient expression efficiency. Differentiation of drug resistant hESC colonies toward primordial germ cells [PGCs] was conducted to confirm stable integration of the transgene
Results: Transient and stable expression suggested a variable potential for different cell lines against transfection. Analysis of parameters that influenced gene transformation efficiency revealed that the vector concentrations from 20-60 Mug and the density of the subjected cells [5×105 and 1×106 cells] were not as effective as the genetic background and voltage rate. The present data indicated that in contrast to the circular form, the linearized vector generated more distinctive drug resistant colonies
Conclusion: Electroporation was an efficient tool for genetic engineering of hESCs compared to the chemical method. The genetic background of the subjected cell line for transfection seemed to be a fundamental factor in each gene delivery method. For each cell line, optimum voltage rate should be calculated as it has been shown to play a crucial role in cell death and rate of gene delivery
RESUMO
Human granulocyte colony-stimulating factor [hG-CSF] can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band, which verified the expression of recombinant protein in lettuce chloroplasts. This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment