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Comparative Study
. 2013 Nov;41(11):934-43.
doi: 10.1016/j.exphem.2013.07.002. Epub 2013 Aug 14.

Comparison of Transduction Efficiency Among Various Lentiviruses Containing GFP Reporter in Bone Marrow Hematopoietic Stem Cell Transplantation

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Free PMC article
Comparative Study

Comparison of Transduction Efficiency Among Various Lentiviruses Containing GFP Reporter in Bone Marrow Hematopoietic Stem Cell Transplantation

Nan Wang et al. Exp Hematol. .
Free PMC article

Abstract

HIV-derived lentiviral vectors have been used widely to transduce non-dividing cells, such as hematopoietic stem cells (HSCs), in the setting of gene therapy. In this study, we screened lentiviral vectors for their ability to drive expression of the murine MHC class II chaperone, invariant chain (Ii) and a GFP reporter. The vectors included T2A vector with T2A-separated Ii and GFP under the same MSCV promoter, dual-promoter vectors with separate promoters for Ii and GFP (called MSCV or EF1a according to the promoter driving Ii expression), and a vector with EF1a driving a fusion of Ii/GFP (called Fusion vector). T2A and MSCV induced the highest levels of Ii and GFP expression, respectively, after direct transfection of 293T cells. All vectors except the Fusion vector drove expression of functional Ii, based on the enhancement of MHC class II level, which is a known consequence of Ii expression. Comparing the vectors after they were packaged into lentiviruses and used to transduce 293T, we found that MSCV and EF1a vectors mediated higher Ii and GFP expression. In ckit(+) bone marrow (BM) cells, MSCV still induced the highest Ii and GFP expression, whereas EF1a induced only robust Ii expression. Regardless of the vector, both Ii and GFP levels were significantly reduced in BM cells compared to 293T cells. When in vivo expression was assessed in cells derived from MSCV-transduced BM-HSCs, up to 80% of myeloid cells were GFP(+), but no Ii expression was observed. In contrast, transplantation of EF1a-transduced BM-HSCs led to much higher in vivo Ii expression. Thus, among those compared, dual-promoter vector-based lentivirus with the EF1a promoter driving the gene of interest is optimal for murine BM-HSC transduction.

Figures

Fig. 1
Fig. 1
Schemes of different pCDH lentiviral vectors. Vectors are named according to the presence or absence of linker (T2A or Fusion) or promoter for Ii in dual-promoter vectors (MSCV or EF1a). Only promoters, transgene (3×Flag tagged Ii) and reporter gene (GFP) are shown. Other elements can be found in the vector maps on System Biosciences website: http://www.systembio.com/lentiviral-technology/expression-vectors/cdna/vector-maps
Fig. 2
Fig. 2
Highest proportion of GFP- and Ii-expressing cells are in MSCV and T2A vector-transfected 293T cells, respectively. A. GFP expression. GFP levels in 293T cells, directly transfected with different vectors, were assessed by FACS. B. Ii (indicated by Flag) expression. Flag levels in the same transfectants were detected by FACS, after intracellular staining with biotin-labeled Flag-specific Ab, followed by allophycocyanin (APC)-conjugated streptavidin. Data shown are representatives of three independent experiments.
Fig. 3
Fig. 3
Highest GFP and Ii levels are induced by dual-promoter vector-derived lentiviruses; Ii expressed by Fusion vector is dysfunctional. FACS analysis of A. GFP expression in 293T cells; B. Ii expression (represented by Flag expression) in 293T cells; C. GFP levels in murine ckit+ BM cells; and D. Ii (Flag) expression in murine ckit+ BM cells transduced with various lentiviruses. Flag levels were detected by intracellular staining prior to FACS analysis, as described in Fig. 2. E. Ii expressed by Fusion vector is not functional. 293T cells expressing I-Ag7 transfected with various vectors were stained with phycoerythrin (PE)-conjugated I-Ag7 specific Ab and then analyzed by FACS. I-Ag7 levels in GFP negative (grey line) and positive (black line) populations were compared. Untransfected cells were used to set the GFP negative threshold (not shown). Data shown are representatives of three independent experiments.
Fig. 4
Fig. 4
In vivo expression of GFP and Ii derived from MSCV or EF1a-transduced HSC. GFP levels (A & B from MSCV; E & F from EF1a) and Ii levels (represented by Flag, C & D from MSCV; G & H from EF1a) were assessed in various cell compartments in blood from NOD mice 4w post-transplantation of HSC. MSCV (A to D) or EF1a (E to H) viruses at MOI=80 were used to transduce donor HSC, which were then transplanted into lethally irradiated recipients. A & C (n=10) and E & G (n=7) summarize data from multiple mice. B & D are representative FACS graphs from one non-transplanted (NT) control and one MSCV lentivirus-transduced mouse. F & H are FACS graphs from another NT control and one EF1a-transduced mouse. Cell markers for each lineage are indicated in parentheses. Note the maximal number on y axis of panel C is 6%.
Fig. 5
Fig. 5
Increased GFP and functional Ii expression 4-5m post-transplantation of EF1a-transduced HSC. GFP (A) and Ii (represented by Flag, B) levels were assessed in various cell compartments in blood from one non-transplanted (NT) control and two EF1a-3FM98A transduced NOD mice. Note the levels of GFP and Flag were not parallel, i.e. EF1a-3FM98A #1 had higher GFP but no Flag expression, whereas EF1a-3FM98A #2 had lower GFP but higher Flag expression. C. I-Ag7 levels in exogenous Ii-expressing (represented by Flag expression, black line) vs. non-expressing populations (grey line) were compared in splenocytes from an EF1a-3FWT transduced mouse (top row of panel C) and an EF1a-3FM98A transduced mouse (bottom row of panel C) 5m post-transplantation. Data shown are representative from two independent experiments.
Fig. 6
Fig. 6
GFP and Ii expression at integration and transcriptional levels. Genomic DNA (gDNA) (A) and complementary DNA (cDNA) from RNA (B) levels for a vector backbone component-WPRE, GFP and Flag were assessed by real time PCR after extraction from 293T cells transduced with various lentiviruses at equivalent volumes using corresponding primers. RRP30 and PPP1CC were used as endogenous controls for gDNA and cDNA respectively. Relative expression levels of corresponding genes were normalized to those derived from T2A-3FWT transduced 293T cells (relative value=1).

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