Skip to main content
. 2014 Jan 21;22(3):607–622. doi: 10.1038/mt.2013.265

Figure 1.

Figure 1

Viral constructs and transgene expression in murine lineage depleted bone marrow and human cord blood CD34+ cells. (a,b) Schematic representation of viral vectors. (a) Retroviral vectors (gRV): the γ-retroviral vector, MND-MFG-ADA (gRV MND ADA) contains the MND retroviral LTRs flanking the wild-type human adenosine deaminase cDNA (hADA) with the Moloney murine leukemia virus packaging region (Ψ) and env splice acceptor fragment (env SA). The gRVSFada/W vector contains hADA driven by SFFV LTR. (b) Lentiviral vectors (LV): All lentiviral vectors contain the enhancer-deleted “SIN” LTR (indicated by the X in the U3 region), the primer binding site (Θ), the HIV-1 packaging signal (Ψ), the central polypurine tract (cPPT), the rev-responsive element (RRE). LV MND ADA contains the MND LTR U3 region enhancer/promoter (MND) driving expression of the hADA cDNA. LV EFS ADA contains the human elongation factor-α gene “short” promoter (EFS) driving expression of the codon-optimized human ADA cDNA (co-hADA) and a Woodchuck Hepatitis Virus posttranscriptional regulatory element (wPRE). The LV EFS GFP vector contains the EFS promoter and green fluorescent protein (GFP). (c,d) Murine ADA−/− bone marrow lineage negative (Lin-) progenitors were transduced for 24 hours with lentiviral or retroviral vectors at a multiplicity of infection (MOI) of 20 after 24 or 72 hours preactivation, respectively (Mean ± SD). ADA expression was analyzed 72 hours after transduction by (c) enzymatic activity assay and (d) Western Blot with whole cell lysates. Mean and standard deviation of ADA activities were calculated from experiments performed with cells obtained from three different ADA−/− donors. (e–g) Human cord blood CD34+ cells were transduced with the vectors at the indicated vector concentration, grown for 2 weeks in myeloid differentiation culture, and assayed for (e) vector copy number (VCN) by quantitative polymerase chain reaction (qPCR) and for (f) ADA enzyme activity by colorimetric assay. (g) The ADA enzyme activity present per VC was calculated. Horizontal bars indicate Mean ± SEM.