Results: 3

1.
<b>Figure 3</b>

Figure 3. From: Optimized Lentiviral Vector Design Improves Titer and Transgene Expression of Vectors Containing the Chicken ?-Globin Locus HS4 Insulator Element.

Reverse transcription kinetic assay for vector cDNA in target cells transduced with the parental and cHS4-insulated lentiviral vectors. 293T cells were transduced with equal amounts (80 ng of p24 Ag) of each vector stock, and DNA, including low-molecular-weight DNA, was subsequently extracted at various time points. (a) The total amount of the reverse-transcribed vector genome was measured in triplicate using a primer-probe set targeted at the human immunodeficiency virus 1 (HIV1) packaging signal sequence. Reverse-transcribed vector genome copy numbers of cells transduced with INS1(+) were significantly lower (P < 0.05), compared to cells transduced with the parental vector at 6, 12, 50, and 144 hours after transduction. (b) 2-LTR circle formation was measured using quantitative PCR. (c) The peak 2-LTR circle formation at 50 hours after transduction. All experiments were performed in triplicate. *P = 0.01. LTR, long-terminal repeat.

Hideki Hanawa, et al. Mol Ther. 2009 April;17(4):667-674.
2.
<b>Figure 2</b>

Figure 2. From: Optimized Lentiviral Vector Design Improves Titer and Transgene Expression of Vectors Containing the Chicken ?-Globin Locus HS4 Insulator Element.

The “reverse-oriented” 0.25-kb core cHS4 insulator coupled with removal of the residual nef sequences increases transgene mRNA. (a) HeLa cells were transduced with low multiplicity of infection (MOI = 0.1) and DNA and total RNA were extracted from the cells. Enhanced green fluorescent protein (EGFP) mRNA was quantified using quantitative reverse transcription–PCR (qRT-PCR) and values, normalized to DNA copy number, are shown plotted against mean fluorescence intensity (MFI). The experiments were performed in triplicate and the error bar indicates the SEM. (b) Schematic diagram of the modified insulated vectors and the positions of the PCR primers used in this experiment. (c,d) The efficacy of the 3′end processing was assessed by qRT-PCR as described in Materials and Methods. The relative and absolute amount of read-through is indicated. The relative read-through value was calculated by dividing the U3-U5 cDNA copy number by the U3-R cDNA copy number. The experiments were performed in triplicate and the error bar indicates the SEM. *P < 0.005.

Hideki Hanawa, et al. Mol Ther. 2009 April;17(4):667-674.
3.
<b>Figure 1</b>

Figure 1. From: Optimized Lentiviral Vector Design Improves Titer and Transgene Expression of Vectors Containing the Chicken ?-Globin Locus HS4 Insulator Element.

Schematic diagrams and gene expression levels of lentiviral vectors containing various cHS4 1.2- or 0.25-kb core insulator configurations. (a) Schematic diagrams of the parental and cHS4 insulator vectors. The 1.2-kb cHS4 insulator was inserted into the SIN-LTR [INS1(+) and INS1(−)] or between enhanced green fluorescent protein (EGFP) cDNA and the 3′LTR [INS2(+) and INS2(−)]. The horizontal arrows indicate the original direction of the insulator elements relative to the chicken β-globin genes. (b–d) HeLa and 293T cells were transduced at a multiplicity of infection (MOI) of 0.1, and K562 cells were transduced with the various vectors at an MOI of 0.4 in triplicate. Mean fluorescence intensity (MFI) and coefficient of variation (CV) values of the EGFP-positive fraction of each cell line are shown with the indicated vectors. The error bar indicates SEM. *P < 0.05, **P < 0.005. (e) Southern blot analysis of DNA from 293T cells transduced with the parental vector or the parental vector containing the 0.25 kb × 2 insulator element, as indicated. P = plasmid, V = vector. DNA was digested with AflII as shown, and probe with an EGFP probe. LTR, long-terminal repeat; MSCV, murine stem cell virus; RRE, rev-responsive element; SIN, self-inactivating.

Hideki Hanawa, et al. Mol Ther. 2009 April;17(4):667-674.

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