RESUMEN
Lentiviral vectors [LVs] are useful vehicle for genetransfer to dividing and non-dividing cells and genetic manipulations. However, the use of lentiviruses in studies requires an accurate titration technique.Quantitative real-time PCR [qPCR] is a sensitive technique for the indication and quantitation of retrovirals particles. In this study, we used the qPCR for lentiviral vector titeration. The puromycin resistance gene as templates for an SYBR green-based real-time qPCR method and detect lentiviral copy number integrated lentiviral DNA. Consequently, this studyshowed that theusing ofantibioticresistance genesviral particles titration maybeefficient with highly accuracy
RESUMEN
Type I diabetes is an immunologically-mediated devastation of insulin producing cells [IPCs] in the pancreatic islet. Stem cells that produce beta-cells are a new promising tool. Adult stem cells such as mesenchymal stem cells [MSCs] are self renewing multi potent cells showing capabilities to differentiate into ectodermal, mesodermal and endodermal tissues. Pancreatic and duodenal homeobox factor 1 [PDX1] is a master regulator gene required for embryonic development of the pancreas and is crucial for normal pancreatic islets activities in adults. We induced the over-expression of the PDX1 gene in human bone marrow MSCs [BM-MSCs] by Lenti-PDX1 in order to generate IPCs. Next, we examine the ability of the cells by measuring insulin/c-peptide production and INSULIN and PDX1 gene expressions. After transduction, MSCs changed their morphology at day 5 and gradually differentiated into IPCs. INSULIN and PDX1 expressions were confirmed by real time polymerase chain reaction [RT-PCR] and immunostaining. IPC secreted insulin and C-peptide in the media that contained different glucose concentrations. MSCs differentiated into IPCs by genetic manipulation. Our result showed that lentiviral vectors could deliver PDX1 gene to MSCs and induce pancreatic differentiation