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Abstract
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Background and Aim: Mesenchymal stem cells (MSCs) are being developed as a new vehicle for de- livering diagnostic and therapeutic elements to a large number of degenerative diseases such as cancers, asth- ma and injuries. Recently, a number of studies have focused on the potency of MSCs for targeted therapy relying on the capacity of MSCs in accepting and deliv- ering Lentiviruses as vehicles and their tropism to the site of infection. Lentiviral vector system is also shown to be a promising tool for incorporating into genomic DNA with high efficiency as the expression of Lentivi- ral vector-mediated transgenic genes are maintained in the cells for long time. Methods: A second generation of lentiviral vector in- cluding pTRH plasmid (pTRH1-mCMV-dscGFP) was used together with two packaging plasmids pSPAX2 (encoding rev, tat, gag/pol) and pMD2G (encoding env proteins) co-transfected into 293 T (HEK) cells using lipofectamine 2000. The virus was harvested by collecting the cell culture supernatant after 24, 48 and 72 h. After filtering the cell supernatant through 0.45 µm filters, the virus was concentrated by centrifuging at 9000 g for 15 min fallowed by a second spin (6000 g, 5 min). The concentrated virus was stored at -20̊ C. Bone marrow derived MSCs were cultured in growth medium and passage 3 was performed in 24-well plates (1×104cells per well). When the MSCs reached to 60 to 80% confluent, they were incubated with the lentivirus for 72 h. After collecting the MSCs with tripsinizing (Tripsin-EDTA 0.25% for 5-6 min), whole genome of transfected MSCs were purified with genomic DNA culture cell purification kit. The PCR reaction was done to detect GFP (Green Flourcent Protein) gene located in the vector. PCR product was assessed with electropho- resis with 1.5% agarose gel. Results: It was showed that MSCs can efficiently be transfected by lentiviral vectors. Since the PCR was performed on the whole genome of MSCs, the virus was inserted in the genome properly. Co
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