Four Viral Vector Mediated Transfection Into Human Dermal Fibroblast In Vitro

Gene therapy is the introduction of genetic material into cells for therapeutic purposes. There are three elements in gene therapy. They are vector, target cells and target gene.Dermal fibroblasts(DFs)are are convenient to obtain and prolific and hard to invert. These characteristics demonstrate that DFs are proper target cells which can be widely used in both gene therapy and gene modification.Recent findings suggest that major breakthroughs in low transfection rate for gene therapy are imminent, thus one of the key points is to use proper vector for gene delivery. The main vectors used in gene therapy include viral vectors and non-viral vectors. As viral vector, RV (retrovirus), Ad (Adenovirus), AAV (Adeno-associated virus) and HSV (herper simplex virus) have been used in clinic which is authorized by Food And Drug Administrtion (FDA) in USA. AAV vectors are prevalent for its stable transfection and can cause almost no pathogen reactions which make it prevalent in gene therapy especially for long-term expression. On the other hand, Adenoviral vectors can easily be obtained at high titers, thus Adenoviral vectors have been widely applied in gene therapy and gene functional study. On the basis of recent study, we investigate the transfection rate of AAV2 and AAV2/1 which inserted the ITRs of AAV2 on the AAV1 as well as Ad5 and Ad5/F35 which is integrated by the fiber nob of Ad35 and the Ad5 vector at different MOIs. The improved vectors may change tissue Addiction in gene delivery.In this investigation, DFs was isolated from human dermal skin which is authorized by related department and primarily cultured. AAV encoding enhanced green fluorescent protein (eGFP) were constructed and transfected in vitro to the human dermal fibroblasts (DFs) at MOI ranging from 1. 0×104 to 1.0×106v·g/ cell .Twenty four hours after the infection, the expression rates of eGFP on cultured DFs were assessed by flow cytometry and the transfected cells were observed by fluorescence microscope. Two AV encoding enhanced green fluorescent protein (eGFP) were constructed and transfected in vitro to the human dermal fibroblast at MOI 10, 25, 50 and 100.Twenty four hours after the infection, the expression rates of eGFP on cultured DFs were assessed by flow cytometry and the transfected cells were observed by fluorescence microscope. The killing effect of the four viruses on infective DFs was assayed respectively by MTT. The assays demonstrated following results:AAV2/1 has no efficiency when transfection DFs. The rate of AAV2 mediated transfection at MOI 105 referred to a peak of (22.16+5.59)% which demonstrated we can obtain gene transfer cells to apply in gene therapy and gene modification using AAV2.Transfection efficiency of Ad5-eGFP and Ad5/F35-eGFP were increased as MOI increased. At MOI 100, the rate of Ad5/F35 mediated transfection is about 90% whereas about 70% of Ad5The results demonstrate Ad5/F35 is the best vetor during the four vectors we investigated and we can obtain gene modification cells for gene therapy using AAV2 with low transfectiong efficiency which means the low efficiency remain a problem on AAV.