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Primers were as follows: forward primer, 5'ACGACGGCAACTACAAGACC3';
reverse primer, 5'GCCATGATATAGACGTTGTGG3'. Amplification was per-
formed in a final volume of 50 µ l, with IQ™ SYBR ® Green Supermix (Bio-Rad,
California, USA), in accordance with the manufacturer's instructions. Amplifica-
tion was carried out over 40 cycles of denaturation at 95°C, annealing at 59°C and
elongation at 72°C. Standard curves for the eGFP sequence were generated by
serial 10-fold dilutions of duplicate samples of the eGFP plasmid in DNA from
untransduced PBMC, with 250 ng of total DNA in each sample. Samples from
animals were run in duplicate, and the values reported correspond to the means
for replicate wells.
statistical Analysis
Paired and unpaired comparisons were performed using non parametric Kruskal
Wallis, Wilcoxon rank and Mann & Whitney tests, respectively, both of which
can be used for the analysis of small samples when normal distribution is uncer-
tain or not confirmed. Tests were performed using StatView 5.01 software (Aba-
cus Concepts, Berkeley, CA).
results
Efficient Transduction of Cynomolgus Macaque CD34 + bone
Marrow cells
We first assessed, in vitro, the efficiency with which a SIVmac251-derived vec-
tor transduced CD34 + hematopoietic cells from macaque bone marrow (BM).
We harvested BM cells from the iliac crests of 12 different animals. CD34 + cell
preparations with a purity of 97% ± 1% were obtained by immunomagnetic pu-
rification. The CD34 + cells were then transduced by coculture for 24 h with the
lentiviral vector (MOI = 100) in medium supplemented with SCF, Flt3-L, IL-3
and IL-6. The vector used (pRMES8) was derived from SIVmac251 and contains
the eGFP reporter gene under control of the phosphoglycerate kinase (pGK) pro-
moter (Figure 1). Transduction efficiency (Figure 2A and 2B), as evaluated by
flow cytometry analysis of eGFP expression at 24 h, was 41% ± 9% on average
(n = 12). After 24 hours of culture with the lentiviral vector, some of the purified
CD34+ cells were cultured for 14 days in semi-solid medium containing SCF,
GM-CSF, IL-3 and EPO to allow the myeloid differentiation of colony-forming
cells (CFC), whereas some cells were cocultured for 35 days on a layer of murine
fibroblasts of the M2-10B4 cell line and were then cultured for 14 days on semi-
solid medium containing SCF, GM-CSF, IL-3 and EPO, for the identification of
long-term culture-initiating cells (LTC-IC). Transduction had no effect on the
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