1. Comparative clonal analysis of reconstitution kinetics after transplantation of hematopoietic stem cells gene marked with a lentiviral SIN or a γ-retroviral LTR vector 
Kerstin Cornils, Cynthia C. Bartholomae, Lars Thielecke, Claudia Lange, Anne Arens, Ingmar Glauche, Ulrike Mock, Kristoffer Riecken, Sebastian Gerdes, Christof von Kalle, Manfred Schmidt, Ingo Roeder, Boris Fehse 
Experimental Hematology 
Volume 41, Issue 1, Pages e3 (January 2013)
DOI: /j.exphem
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

2. Figure 1
Vector design and experimental set-up. (A) The SF91-eGFP vector was described previously [26]. The Lenti-SF-eGFP vector was constructed by cloning the SFFV-promoter element from the SF91 into the LeGO-G2 vector [28]. (B) Viral supernatants of the two vectors were used to transduce lineage-depleted bone marrow cells from male CD45.2 mice. Transduced cells were transplanted into lethally irradiated female CD45.1 recipients (n = 10). Animals were observed for 7 months. During that observation time blood samples were taken every 4 to 5 weeks.
Experimental Hematology  , e3DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

3. Figure 2
FACS analysis of freshly transduced and peripheral blood cells to detect eGFP expression. (A) Analysis of transduced cells by flow cytometry for expression of eGFP and the stem cell marker c-kit (phycoerythrin-coupled antibody) 48 hours after transduction. As evident, transduction efficiency in the SF91-transduced cells was approximately 4 times higher in comparison to Lenti-SF–transduced cells. (B) Percentage of eGFP-expressing cells in whole-blood samples taken at indicated time points post-transplantation. Relative numbers of eGFP-expressing cells remained stable over the observation time in both experimental groups (n=4 randomly chosen animals per group).
Experimental Hematology  , e3DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

4. Figure 3
Mean numbers of clusters per 100,000 transplanted, transduced cells in the peripheral blood of individual mice at different time points. Numbers of clusters were determined after processing the obtained sequences with a cluster algorithm (compare Materials and Methods). Numbers of clusters were calculated for individual animals per 100,000 transplanted, eGFP-positive transduced cells based on the initial transduction rate of 0.74 for SF91 and 0.18 for Lenti-SF. Data represents mean values for four animals per group.
Experimental Hematology  , e3DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

5. Figure 4
Clonal composition of hematopoiesis over time. Kinetics of relative contribution of individual sequence clusters (also referred to as clones) to hematopoietic reconstitution after transplantation are shown for the two vector groups. Data shown reflect a summarized analysis for each of the four animals per group. Relative clone sizes were adjusted to the total sequences obtained for each individual mouse at each data point.
Experimental Hematology  , e3DOI: ( /j.exphem )
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6. Figure 5
Mean numbers of clones/clusters containing insertion sites in the proximity of retrovirally tagged cancer genes and their abundance in the blood of mice over time. Cluster sequences were blasted against the mouse genome; genes in a window of ±250 kb were identified and analyzed for their presence in the RTCGD [37]. (A) Mean numbers of RTCGD clusters in relation to total cluster numbers are depicted for the four animals per vector group and the different time points. (B) Read counts for the RTCGD-clusters [as in (A)] were set in relation to the total reads for the four individual mice at all time point; depicted are the means for both groups; whiskers indicate the standard error; p values indicate significant differences between the two groups, n.s. = not significant.
Experimental Hematology  , e3DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

7. Figure 6
Comparative quantitative analysis of LAM-PCR amplicon sequence counts and clone-specific qPCRs. We directly compared quantitative analysis of clonal contribution as determined by LAM-PCR and NGS (relative read counts/cluster numbers) and integration-specific qPCR (copy ratio). Two different examples of outcomes are shown. (A) Integration-specific quantitative real-time PCR for an integration found in mouse I.4.7 was established. Comparison of qPCR data with quantitative cluster analysis revealed a very good correlation. In line, analyses of the restriction sites of the used enzymes near the insertion locus showed a good accessibility at least for Tsp509I. (B) On the contrary, only weak correlation of integration-specific qPCR and quantitative cluster analysis based on 454 sequencing was found for an integration site from mouse I.4.5. As indicated, restriction sites for both enzymes used for LAM-PCR were relatively distant from the insertion, which strongly impairs detection by LAM-PCR [24].
Experimental Hematology  , e3DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions