Abstract
Gene expression studies and gene therapy require efficient gene delivery into cells. Different technologies by viral and non-viral mechanisms have been used for gene delivery into cells. Small gene vectors transfer across the cell membrane with a relatively high efficiency, but not large genes or entire loci spanning several kilobases, which do not remain intact following introduction. Previously, we developed an efficient delivery system based on herpes virus simplex type 1 (HSV-1) amplicons to transfer large fragments of DNA incorporated in human artificial chromosome (HAC) vectors into the nucleus of human cells. The HSV-1 amplicon lacks the signals for cleavage and replication of its own genome, yet each amplicon has the capacity to incorporate up to 150 kb of exogenous DNA. In this study, we investigated whether the capacity of gene delivery could be increased by simultaneously introducing multiple HSV-1 modified amplicons carrying a gene expressing HAC vector into cells with the aim of generating a single artificial chromosome containing the desired genes. Following co-transduction of two HSV-1 HAC amplicons, artificial chromosomes were successfully generated containing the introduced genes, which were appropriately expressed in different human cell types.
Highlights
Gene expression vectors are useful for monitoring expression in human cells and complementation studies of genetic disorders for gene therapy
The human induced pluripotent stem cell (hiPS) and hESc were grown on inactivated mouse embryo fibroblasts medium composed of DMEM-F/12(Sigma Aldrich) medium supplemented with 20% (v/v) KnockOut Serum Replacement (ThermoFisher Scientific), 0.1 mM nonessential amino acids (Sigma Aldrich), 1x GlutamaxTM-1 (Sigma Aldrich), 0.1 mM β-mercaptoethanol, 10 ng/ml basic fibroblast growth factor (ThermoFisher Scientific) and 1% (v/v) penicillin and streptomycin
Following co-transduction of two herpes virus simplex type 1 (HSV-1) human artificial chromosome (HAC) amplicons, one with 17α DNA and RFP (17 RFP) and the other with 21α DNA and GFP (21 GFP) into HT1080 cells at multiplicity of infection (MOI) 1 and 5, the cells expressing GFP + RFP were monitored after 24 h and compared to those expressing either GFP or RFP alone (Fig. 2A)
Summary
Gene expression vectors are useful for monitoring expression in human cells and complementation studies of genetic disorders for gene therapy. Different types of vectors have been utilised for efficient and intact transfer across the cell membrane. Non-viral methods have included transfer of genes in conjunction with liposomes, peptides, polymers, transposases or naked DNA introduced mechanically via electroporation, microinjection, sonoporation, magnetofection and gene gun delivery. Adenovirus adenoassociated virus, lentivirus, alphavirus, baculovirus and herpes virus [1]. Genomic clones as bacterial or yeast artificial chromosomes (BAC, YAC) were constructed to incorporate whole loci up to several hundred kilobases in size, delivery of these larger vectors into cells was inefficient due to ectopic DNA degradation following transfer [2]
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