Volume 9, Issue 2, Pages 209-217 (February 2004) Lentiviral Vectors Efficiently Transduce Quiescent Mature 3T3-L1 Adipocytes  Françoise Carlotti, Merlijn Bazuine, Tuija Kekarainen, Jurgen Seppen, Philippe Pognonec, J.Antonie Maassen, Rob C Hoeben  Molecular Therapy  Volume 9, Issue 2, Pages 209-217 (February 2004) DOI: 10.1016/j.ymthe.2003.11.021 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Transduction efficiency in 3T3-L1 adipocytes after adenovirus- and lentivirus-mediated gene transfer. Mature adipocytes were exposed (A) to adenoviral vectors containing a GFP reporter gene under the control of a CMV promoter at m.o.i. of 100, 400, or 1000 pfu/cell or (B) to lentiviral vectors containing GFP under a PGK promoter at 200 and 400 ng p24 per 105 cells. (C) Transductions were performed as a control with adenoviruses (top) at m.o.i. of 5 in HeLa cells (picture after 2 days) and with lentiviruses (bottom) at 40 ng p24 per 105 cells in 293T cells (picture after 3 days). m.o.i., multiplicity of infection. Two different magnifications were used in A and B (left and right). Molecular Therapy 2004 9, 209-217DOI: (10.1016/j.ymthe.2003.11.021) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Schematic representations of the lentivirus vectors. (A) The constructs are all derivatives of the third generation of HIV-1 lentivirus vectors. The top depicts various elements in the self-inactivating lentiviruses as described in the text. The enhancer region contained in the 3′ LTR is deleted. The internal promoter drives expression of the gene of interest (X). Also indicated are the positions of the Rev-responsive element (RRE), the central polypurine tract (cPPT), and the posttranscriptional regulatory element (PRE). The bottom depicts the structure of the integrated provirus and illustrates the single transcript (arrow) that is derived from it. (B) Schematic outline of the three helper plasmids that were used to produce the vector stocks. These plasmids provide the messengers encoding the gag–pol and the rev from HIV-1 and the envelope glycoproteins from the vesicular stomatitis virus (VSV-G). The promoters used in these constructs are derived from the human cytomegalovirus (CMV) and Rous sarcoma virus (RSV), the polyadenylation signals from simian virus 40 (SV40) and the human β-globin gene (hβG), as indicated. Molecular Therapy 2004 9, 209-217DOI: (10.1016/j.ymthe.2003.11.021) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Insulin-induced glucose uptake assay. 3T3-L1 mature adipocytes were exposed (A) to Ad-CMV-Luc at m.o.i. of 100, 400, or 1000 pfu/cell or (B) to LV-PGK-GFP at 80 ng p24 per 105 cells. Luciferase activity was measured as a control of the transduction efficiency with the adenovirus, and GFP expression was determined by microscopy to control for lentiviral transduction. Transduced cells were stimulated by insulin (100 nM) for 15 min and assayed for intracellular 2-deoxy-d-[14C]glucose (2-DOG) uptake. Values are means of three to six independent experiments and experiments were repeated at least two times. Values are given in pmol 2-DOG/min · mg protein. Molecular Therapy 2004 9, 209-217DOI: (10.1016/j.ymthe.2003.11.021) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Confocal microscopy of mature adipocytes transduced by lentiviruses. (A) 3T3-L1 mature adipocytes (B and C) were transduced by lentiviruses expressing a PGK-GFP cassette (120 ng p24 per 105 cells) and (D) stained by Nile red dye. (E) An overlay of GFP expression in the cytoplasm and the nucleus and Nile red staining specifically in the lipid droplets. Molecular Therapy 2004 9, 209-217DOI: (10.1016/j.ymthe.2003.11.021) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Comparison transfection/lentivirus transduction. (A) 3T3-L1 preadipocytes were transduced by CMV-GFP lentivirus at 20, 40, 80, or 200 ng p24 per 105 cells or transfected with 0.1, 0.25, 0.5, or 1 μg of pLV-CMV-GFP plasmid using JetPEI reagent. (B) 293T cells were infected in a similar way (4, 10, 20, 40, 80, or 200 ng p24 per 105 cells by lentiviruses) or transfected using the CaPO4 method with the same amounts of DNA. GFP-positive cells were determined by UV microscopy, and percentages were defined by FACS analysis after 3 days. Experiments were performed in 24-well-plates with 1 μg DNA/well in total. pLV-CMV-GFP plasmid was used, and adjustments to obtain equal amounts of DNA were done with pLV-CMV vector. Molecular Therapy 2004 9, 209-217DOI: (10.1016/j.ymthe.2003.11.021) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Detection of integrated provirus and stability of expression. (A) 3T3-L1 adipocytes were transduced by LV-CMV-GFP lentivirus (200 ng p24 per 105 cells) or mock transduced (0 ng p24 per 105 cells; top). Four days after transduction, genomic DNA was extracted as described and assayed using B2-nested PCR analysis as described under Materials and Methods. The bottom left corresponds to the PCR products from the B2-vector reaction (PCR 1), and the bottom right corresponds to the nested PCR (PCR 1 + 2) and the control reactions (PCR 2) with pLV-CMV-GFP plasmid (C+) and unamplified genomic DNA. (B) Transduced (40 ng p24 per 105 cells) or untransduced (Mock) 3T3-L1 preadipocytes were differentiated into mature adipocytes. Transduction efficiency was followed by UV microscopy after 4 days (Before Differentiation) and after 15 days of differentiation (After Differentiation). Molecular Therapy 2004 9, 209-217DOI: (10.1016/j.ymthe.2003.11.021) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions