Lentivirus Gene Engineering Protocols PDF Download

With their advantages including wide tropism, high efficiency in gene transfer to both dividing and non-dividing cells, and stable and long-term expression of transgenes, lentiviral vectors have been attractively used for genes therapies and widely used for basic biomedical researches where gene transfer is required. As expression of multiple genes from the same target cell is required in such applications, this research work focused on designing novel multicistronic lentiviral vectors to develop gene therapy of diabetes through regulated insulin delivery from skin cells and live cell arrays for analyzing gene expression in a high-throughput and real-time manner.^Specifically, first, lentiviral vectors were engineered to produce a fusion protein between the furin cleavable proinsulin gene and the self-dimerization mutant of FK506-binding protein to yield bioactive insulin in keratinocytes that could be released following exogenous administration of a small organic molecule, rapamycin. The engineered keratinocytes retained normal morphology and grew similar to lentiviral-treated control cells. Epidermal keratinocytes in culture and in stratified bioengineered epidermis released insulin within 30 min after addition of rapamycin, and secretion slowed or stopped within 2-3 hours after removal of the inducer. When the cells were implanted into athymic mice that were rendered diabetic with streptozotocin, insulin was detected in the plasma within 1 hr after addition of rapamycin. Concomitantly, glucose decreased to normal levels even in diabetic animals suffering severe hyperglycemia. Repeated rapamycin administration yielded similar results.^These experiments provide proof-of-concept that insulin released from the skin in a regulatable manner can be an effective treatment for diabetic patients. Second, a lentiviral vector carrying two transcriptional units was designed to reach independent and high level dual-gene expression. The two transcriptional units were separated by polyadenylation, terminator and insulator sequences in order to eliminate promoter interference existing between the transcriptional units. With this design, the expression level of both genes was as high as that yielded from lentiviral vectors containing only a single transcriptional unit. Similar results were observed with several promoters and cell types including epidermal keratinocytes, bone marrow mesenchymal stem cells and hair follicle stem cells.^Notably, this research work also demonstrated quantitative dynamic monitoring of gene expression in primary cells with no need for selection protocols suggesting that this optimized lentivirus may be useful in high-throughput gene expression profiling studies. Last, using the novel double-promoter lentiviral vector scalable live-cell microarrays were developed to measure gene expression dynamics in a real-time and high-throughput manner. To this end, dual-promoter lentiviral vectors were prepared harboring a transcriptional regulatory element encoding for green fluorescence protein to monitor cell activation in response to exogenous stimuli and a constitutive promoter driving red fluorescence protein for internal signal normalization.^Lentivirus preparations were immobilized in a microarray format and after transduction on the array surface target cells were treated with cytokines and interrogated in real-time using automated fluorescence microscopy, providing rich dynamic information over a period of several days. Data normalization by red fluorescence intensity eliminated errors due to spot-to-spot variability in transduction efficiency or changes in cell proliferation upon cytokine treatment. These results suggest that the LVA can monitor gene expression in real-time and high-throughput manner thereby providing a useful tool for quantitatively measuring gene expression dynamics and deciphering gene regulatory networks.