Figure 1.

Integrase-defective lentiviral vectors (IDLVs) infect murine bone marrow–derived cells (BMDCs) and induce expression of endogenous mRNA, and transduce HT1080 cells with minimal integration. Murine Lin–/Sca1+ cells were infected with ILV3-RPE65 (A) or IDLV3-RPE65 (B) on RetroNectin by centrifugation for 2 h at 150 g prior to harvest for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. HT1080 cells were infected with IDLV3-GFP (C) or ILV3-GFP (D) with polybrene for 16 h and cultured for 1–12 days with green fluorescent protein (GFP) expression measured each day. Uninfected cells were used as a negative control. For the GFP assays, cells stably expressing GFP were used as a positive control. (A) Expression of RPE65 mRNA from ILV3-RPE65 was ∼30-fold higher than in untreated cells. Cells also expressed the endogenous Rpe65 and Cralbp mRNA approximately six- and fourfold higher than uninfected cells, respectively (p < 0.05). (*p < 0.05; n = 9). (B) Expression of RPE65 mRNA from ILV3-RPE65 was ∼28-fold higher than expression in the negative control (p < 0.05). Expression of RPE65 mRNA from IDLV3-RPE65 was approximately eightfold higher than in the negative control (p < 0.05). Expression of RPE65 was significantly lower from IDLV3-RPE65 than ILV3-RPE65 (p > 0.05). (*p < 0.05; n = 3). (C) The percentage of cells expressing GFP was measured each day. Between day 1 and day 6, expression of GFP reduced from ∼13% to ∼6% (p < 0.05). Expression further reduced between day 6 and day 12 from ∼6% to ∼1% (p < 0.05). (*p < 0.05; n = 3). (D) The percentage of cells expressing GFP was measured each day. Between day 1 and day 4, GFP expression increased from ∼64% to 82% (p < 0.05) before stabilizing at ∼75% through day 12. (*p < 0.05 [standard error of the mean (SEM)]; n = 3). All statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test.