1. Volume 6, Issue 1, Pages 19-29 (July 2002)
Biodistribution and Toxicity Studies of VSVG-Pseudotyped Lentiviral Vector after Intravenous Administration in Mice with the Observation of in Vivo Transduction of Bone Marrow 
Dao Pan, Roland Gunther, Weiming Duan, Steve Wendell, William Kaemmerer, Tal Kafri, Inder M. Verma, Chester B. Whitley 
Molecular Therapy 
Volume 6, Issue 1, Pages (July 2002)
DOI: /mthe
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Real-time PCR to quantitate both GFP transgene and Apob internal control in a single-well duplex reaction. The standards contained different amounts of GFP (ranging from 0.001% to 100%) but the same amount of total genomic DNA (1 μg). (A) Amplification plots for GFP (top) and Apob (bottom) that derived from the same set of reaction wells (12 wells of six standard samples). The horizontal black line represents the target fluorescence threshold (positioned at 0.2 for FAM and 0.1 for VIC). (B) Comparison of same-well threshold cycle numbers (Ct) for both GFP and Apob, which were derived from a series of five 1:10 dilutions of the one copy GFP/cell standard. Each dilution was assayed in triplicate with standard deviation < 0.3 cycles for all points. (C) Comparison of two GFP standard curves generated from standard samples. The Ct-GFP was either normalized by in-well Apob reading or by equal OD reading. Normalized threshold cycle number (norCt) is plotted against GFP transgene frequency (dashed line). Linear regression analysis is shown as a solid line. (D) Reproducibility of standard curve. Each bar represents the mean of 20 individual PCR reactions (of 10 PCR runs) from the same standard sample. The error bar indicates the standard deviation for each point.
Molecular Therapy 2002 6, 19-29DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Organ distribution of GFP transgene in short-term animals. Four days after tail-vein injection of 100 μl of HR'cmvGFP vector, six BALB/c mice (TrM for male and TrF for female) were analyzed for GFP transgene frequency in 10 organs. Samples from control animals were processed together with treated mice, and GFP transgene was found to be undetectable to in all organs from control mice. Organs were collected in the order from gonad to bone marrow (BM) (as shown in the figure from right to left). Real-time QPCR for each sample was carried out in triplicate with SD < 0.5% of norCt. Standard samples were amplified every time with each individual PCR run.
Molecular Therapy 2002 6, 19-29DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Transgene frequency in whole blood from treated mice. Blood was collected periodically on days 4, 11, 25, and 40 after injection. QPCR assay was done in triplicate using a variable amount of DNA (ranging from 0.05 to 1 μg/well). Solid short lines represent the mean of samples from several animals (3 to 11) collected at the same time point. Standard samples were amplified every time with each individual PCR run. GFP was undetectable in blood collected from control animals.
Molecular Therapy 2002 6, 19-29DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
Flow-cytometric analysis for GFP expression in blood 40 days after injection. Dot plots of 10,000 gated events are shown. Percentage of events calculated in the right-side regions is indicated in control mice ContM3 (A) and in treated mouse TrM7 (B). Cells were stained for B-cell-presenting marker with rat anti-mouse CD45R/B220, and for T cells with hamster anti-mouse CD3e monoclonal antibodies.
Molecular Therapy 2002 6, 19-29DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Transgene expression in liver from mice 4 days after injection. The treated mice were injected i.v. with 2 × 107 TU (titered on 293 cells) vector stock into the tail vein over 3–6 seconds. For analysis, sodium Nembutal-sedated mice received transcardial perfusion with PBS and 4% formaldehyde-PBS. Excised liver was cryosectioned and stained with anti-GFP antibody to detect transgene expression. Images are cross sections of the liver of a treated mouse, magnifications: (A) ×100, (B) ×600. Most GFP-staining cells (reddish-brown staining of cytoplasm) are hepatocytes.
Molecular Therapy 2002 6, 19-29DOI: ( /mthe )
Copyright © 2002 American Society for Gene Therapy Terms and Conditions