1. Volume 12, Issue 6, Pages 1206-1216 (December 2005)
Stable Transduction of Primary Human Monocytes by Simian Lentiviral Vector PBj 
Michael D. Mühlebach, Nina Wolfrum, Silke Schüle, Ulrich Tschulena, Ralf Sanzenbacher, Egbert Flory, Klaus Cichutek, Matthias Schweizer 
Molecular Therapy 
Volume 12, Issue 6, Pages (December 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Schematic representations of SIVsmmPBj1.9- and HIV-1 (clone NL4-3)-derived vectors. Only the relevant portion of each plasmid is shown. Coding regions of all viral proteins except env are indicated. The functional expression of the env genes is prevented by deletion of large portions of the region encoding the surface unit. The splice donor site (SD), the Rev-responsive element (RRE), and the packaging signal (Ψ) are indicated. In the egfp-transferring constructs pPBjΔE-EGFP, pPBjΔEΔnef-EGFP, and pNL4-3ΔE-EGFP, the egfp gene is expressed from the CMV promoter (PCMV) located downstream of the RRE. Sites of point mutations in the nef ORF of pPBjΔEΔnef and pPBjΔEΔnef-EGFP are indicated by a star.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Analysis of cell cycle arrest. GHOST/CD4-CXCR4 cells or α-1 diploid fibroblasts were arrested in different phases of the cell cycle as indicated. Arrested cells were stained with propidium iodide for DNA content and analyzed by flow cytometry. The relative proportion of cells in the respective phase of the cell cycle is indicated.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
α-1 diploid fibroblasts transduced at different stages of the cell cycle. Cells were arrested in G1/S by aphidicolin treatment (10 μM) or in G0 by starvation and infected with env-deleted vectors pseudotyped with VSV-G as indicated. 100-fold original magnification. Scale bar, 200 μm. (A) Detection of gene transfer by immunostaining of cells for expression of viral genes 3 days after transduction using cross-reactive serum of HIV-2-positive or HIV-1-positive individuals for SIVsmmPBj- or HIV-1-transduced cells, respectively. Transduction by MLV control vectors was scored by X-Gal staining. (B) Detection of gene transfer using egfp-transferring vectors by expression of EGFP 3 days after transduction.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Transduction of primary human monocytes by SIVsmmPBj- or HIV-derived vectors. (A) DNA/RNA content of monocytes at different days after isolation. FACS analysis of cells stained with Pyronin Y and 7-amino-actinomycin D is shown. Percentages of monocytes in G0 phase are indicated. (B) EGFP expression in primary human monocytes and monocyte-derived macrophages transduced at the indicated day after isolation of monocytes and visualized 1 week after transduction. Results obtained with cells from one representative donor of six are shown. Vectors used for transduction were pseudotyped with VSV-G and are indicated on the left. Scale bar, 100 μm. (C) Percentage of EGFP-expressing monocytes and monocyte-derived macrophages transduced at different days after isolation using various lentiviral vectors and assayed 1 week later. Means of four different donors from one representative of three independent experiments are shown. (▪) SIVPBj-EGFP(VSV), (□) SIVPBjΔnef-EGFP(VSV), (▴) HIVNL4–3-EGFP(VSV), (♦) HIVHR´-EGFP(VSV). (D) Differentiation of MACS-purified monocytes into macrophages as indicated by decreasing CD86 surface expression analyzed by flow cytometry. Percentage of CD86-positive cells from one representative donor is shown. (E) Expression of EGFP and dendritic cell marker CD1a of monocytes transduced with the vectors indicated and subsequent induction of DC differentiation for 4 days. Percentages of cells within each quadrant are shown from one representative experiment.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Integration of SIVsmmPBj-derived proviral DNA demonstrated by Southern blot analysis. (A) Localization of the hybridization probe (thick line) on the SIVsmmPBj1.9 vector genome. Arrows indicate XmnI restriction sites and the lengths of XmnI restriction fragments are given. (B) Schematic representation of different viral DNA forms occurring after reverse transcription in lentivirus-infected cells. Arrows indicate XmnI restriction sites. Specific restriction fragments detected by the probe are indicated by thick lines and their sizes are given. Size of LTR is 0.8 kb. (C, D) Southern blot analysis of total DNA prepared from SIVsmmPBj-transduced cells arrested in different phases of the cell cycle and cleaved by XmnI. Predominance of the 1.9-kb fragment indicates chromosomal integration of provirus [25]. (C) DNA from GHOST/CD4-CXCR4 cells. Lanes 1, reference, PM1 cells infected with SIVsmmPBj1.9-wt virus; 2, proliferating; 3, G1/S arrested; 4, G0 arrested; 5, mock. (D) DNA from human diploid fibroblasts. Lanes 1, reference, PM1 cells infected with SIVsmmPBj1.9-wt virus; 2, proliferating; 4, G0 arrested; 5, mock. (E) Southern blot analysis of total DNA prepared from primary human monocytes. (F) Densitometric analysis of the autoradiogram shown in (E). (Open bar) Fraction of viral DNA in the linear unintegrated, (diagonal stripes ) one-LTR-circle, (vertical stripes) two-LTR-circle, or (solid bar) integrated form. (E, F) Lanes 1, reference, PM1 cells infected with SIVsmmPBj1.9-wt virus; 2, monocytes transduced with SIVPBj-EGFP(VSV); 3, monocytes transduced with SIVPBjΔnef-EGFP(VSV); 4, mock.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Integration of vector DNA and presence of two-LTR circles in transduced monocytes demonstrated by PCR. Monocytes or HT1080 cells used as controls were transduced with HIV-1- or SIVPBj-derived vectors. 7 days posttransduction, DNA was isolated and analyzed by PCR using primers specific for the respective vectors. Agarose gels contain amplification products of monocytes (m) or HT1080 (HT) cells transduced with the vectors indicated. M, mock-transduced cells. (A) Two-step Alu-PCR for detection of integrated vector DNA. Lanes a, amplification products after two rounds of amplification; b, amplification products of virus-specific PCR (second round) without previous Alu-PCR. Time of transduction of monocytes was 1 (d1) or 4 days (d4) after purification. As a control, a fragment of the human β-actin gene was amplified (β-Actin). (B) PCR for detection of two-LTR circles. Time of transduction of monocytes was 1 day after purification.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions