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. 2018 Feb 1;29(1):44–59. doi: 10.1089/hgtb.2017.082

Figure 2.

Figure 2.

Optimization of the use of RetroNectin for infection of BMDCs. Murine (Lin–/Sca1+) or human (CD34+) BMDCs were infected at a multiplicity of infection (MOI) of 50 with ILV3-RPE65 or IDLV3-RPE65 with polybrene (8 μg/μL), protamine sulfate (10 μg/μL), or RetroNectin (2, 4, or 12 μg/μL; or 2, 4, or 12 μg/cm2), or a combination of polybrene and RetroNectin (2 μg/cm2). Cells were infected for 12 h and harvested for qRT-PCR analysis at 16 h. (A) Human RPE65 mRNA levels increased ∼25-fold (murine BMDCs) and ∼23-fold (human BMDCs) with ILV3-RPE65 infection on RetroNectin. Similar levels were observed with polybrene: 24-fold (p < 0.05) in the murine model and 23-fold (p < 0.05) in the human model. Protamine sulfate was less effective, with around an eightfold (p < 0.05; murine) and a ∼10-fold (p < 0.05; human) increase in human RPE65 mRNA. (B) With IDLV3-RPE65, human RPE65 mRNA increased ∼15-fold (p < 0.05; murine) and ∼14-fold (p < 0.05; human) with RetroNectin, 13-fold (p < 0.05; murine) and 14-fold (p < 0.05; human) with polybrene, and around sixfold (p < 0.05; murine) and fivefold (p < 0.05; human) with protamine sulfate. (C) Human RPE65 mRNA levels were increased ∼29-fold (murine) and ∼28-fold (human) BMDCs infected with RetroNectin, 27-fold (p < 0.05; murine) and 28-fold (p < 0.05; human) with polybrene, and ∼21-fold (p < 0.05; murine) and ∼20-fold (p < 0.05; human) with RetroNectin and polybrene in combination. (D) Human RPE65 mRNA levels were increased ∼16-fold over control in human BMDCs infected with ILDV3-RPE65 using RetroNectin, 16-fold (p < 0.05) with polybrene, and ∼13-fold (p < 0.05) with RetroNectin and polybrene in combination. (E) Murine and human BMDCs infected with ILV3-RPE65 in the presence of 2 μg/cm2 RetroNectin expressed human RPE65 mRNA mRNA 27- and 28-fold over control (p < 0.05), respectively. Expression was not increased in either cell type with an increase in RetroNectin concentration to 4 or 12 μg/cm2. (F) Murine and human BMDCs infected with IDLV3-RPE65 in the presence of 2 μg/cm2 RetroNectin expressed human RPE65 mRNA 13-fold over control (p < 0.05) in both cell types. Expression was not increased in either cell type with an increase in RetroNectin concentration to 4 or 12 μg/cm2. (G) Murine and human BMDCs infected with ILV3-RPE65 in the presence of 2 μg/cm2 RetroNectin used to coat one plate expressed human RPE65 mRNA 23- and 24-fold over control (p < 0.05), respectively. Levels did not increase or decrease in cells infected with ILV3-RPE65 in the presence of 2 μg/cm2 RetroNectin used to coat two or three plates. In contrast, where 2 μg/cm2 RetroNectin was used to coat a fourth plate, RPE65 mRNA levels were only seven- and eightfold over control (p < 0.05) in murine and human cells, respectively. (H) Murine and human BMDCs infected with ILV3-RPE65 on 2 μg/cm2 RetroNectin-coated plates expressed human RPE65 mRNA 24- and 25-fold over control, respectively (p < 0.05). Cells transduced with media containing 2, 4, or 12 μg/μL RetroNectin did not significantly express RPE65 mRNA. (*p < 0.05 [SEM]; n = 3). All statistical significance was calculated using one-way ANOVA followed by Tukey's multiple comparisons test.