Lenti x high titer lentiviral packaging system

TRIPZ shRNA plasmid against PTEN was purchased from Thermo Scientific (Clone ID: V3THS_312158: 5'-GGAAAGAATCAAGGAGG-3', Loughborough, UK). Lentiviral production was performed per the supplied protocol using the Lenti-X high-titer lentiviral packaging system in Lenti-X 293T cells (Clontech, Mountain View, USA). After 48 hours incubation, lentivirus-containing supernatant was harvested and applied to MeWo cells at an MOI of 1 or 3. Once stable in culture, puromycin selection was initiated at a concentration of 1μg/ml. To induce shRNA expression, doxycycline was added to culture medium at a concentration of 0.5-1μg/ml and replaced every 72 hours. Confirmation of reduced transcript production by qRT-PCR was performed on an Applied Biosystems 7500 FAST cycler using Qiagen PCR primers against PTEN [Qiagen Quantitect primers (HS_PTEN_4_SG and HS_GAPDH_1_SG)]. Absence of PTEN protein expression was confirmed on Western blot.

For the noninvasive assay of ectopic lesion growth in mice with endometriosis, the firefly luciferase gene was cloned into the pSMPUW-Hygro construct (Cell Biolabs, catalog number: VPK-214). Lentivirus carrying luciferase gene were generated 293 T cells by transfection with pSMPUW-Hygro containing luciferase gene and the Lenti-X high-titer lentiviral packaging system (ClonTech, catalog number: 631278). The recombinant lentivirus titer was measured using Lenti-X™ GoStix™ Plus (ClonTech, catalog number: 631280). IHEECs and IHESCs were transduced with lentiviral vectors carrying the luciferase expression cassette with TransDux MAX™ (System Bioscience, catalog number: LV860A-1). Luciferase-labeled IHEECs and IHESCs were then selected in the presence of 300 µg/ml hygromycin. The luciferase gene expression in these recombinant cells was validated using a luciferase activity assay kit (Promega, catalog number: E1980). All these recombinant cells were maintained with DMEM/F12 supplemented with 10% FBS and penicillin/streptomycin under drug selection.