Case Studies

In this study performed at the VVPP, a 293T stable cell line for the overexpression of the human angiotensin converting enzyme 2 (hACE2) was initially generated (293T cells do not express the hACE2 gene) using a VSV-G envelope-based lentivirus (1). Then, we created a pseudotyped lentivirus for coronavirus by replacing the VSV-G glycoprotein with the Spike2 wild type glycoprotein and three of its different mutated forms. To monitor the transduction efficacy of our viruses, we made the viruses to express the DsRed protein (2). As depicted in the above figure (3) only the VSV-G lentivirus could enter wild-type 293T cells, but not the Spike2 pseudotyped lentivirus, which requires the hACE2 receptor. Thus, both VSV-G and Spike2 viruses could enter the 293T-hACE2 stable cell line. Interestingly, the mutated forms of the wild type Spike2 glycoprotein could confer to the related pseudotyped lentiviruses a significant higher transduction capacity. This study was performed in the context of the screening for monoclonal antibodies antagonizing coronavirus.

At the VVPP we created an AAV serotype 8 for iCre and GFP expression, under the control of the liver specific promoter (Thyroxine-binding globulin) TBG, were injected into loxP mice for the gene of interest (GOI). 3 weeks and 16 weeks after injection, the liver was harvested and investigated for gene expression analysis. As shown in this figure, a significant knock-down for the GOI was specifically obtained in the liver after 3 weeks and maintained for 16 weeks.

At the VVPP we created an AAV serotype 8 for GFP expression and an Adenovirus (Ad) for RFP365 expression, both under CMV promoter. The viruses were locally injected into interscapular brown adipose tissue (iBAT). 2 weeks (for AAV) and 1 week (for Ad) later, the whole iBAT was initially inspected for the expression of the related fluorescent protein. Then, after separation of the stromal vascular fraction (SVF) from the mature fraction (MF), it was evident that AAV serotype 8 could allow to specifically target the MF, while the Ad was less specific by targeting both SVF and MF.

At the VVPP, a recombinant murine stem cell virus (MSCV) for the co-expression of Puromycin (PURO) and GFP was packaged into Phoenix-ECO cells. After purification and titration, the virus efficacy was tested on different mouse adipocytes. As shown in the above figure, transduction of mature 3T3-L1 cells, as well as immortalized white and brown adipocytes, resulted in a significant expression of GFP.

At the VVPP we generated an all-in-one lentivirus-based Tet-On inducible expression system as indicated in the above figure. Briefly, the human phosphoglycerate kinase 1 promoter (hPGK) drives the expression of the reverse tetracycline-controlled TransActivator (rtTA), while the 3rd generation Tet-responsive promoter is ready to induce the expression of the downstream cloned gene (GFP in this case, but it could also be any gene of interest, overexpression or a mirR30-based shRNA). Furthemore, the cytomegalovirus (CMV) promoter drives the expresison of mCherry (to monitor trasduction efficacy) and Puromycin (for the generataion of stable cell lines). Upon Doxycycline treatment on the generated stable cell line, rtTA could bind to the TRE3GS promoter to induce the expresison of the downstream gene, as indicate in the above picture by the appearance of GFP.