Reconstitution of the Myeloid and Lymphoid Compartments after the Transplantation of Autologous and Genetically Modified CD34 Bone Marrow Cells, Following Gamma Irradiation in Cynomolgus Macaques - Genetic Engineering: Recent Developments in Applications - page 131

Table 1. Reconstitution with transduced autologous CD34 + cells in irradiated cynomolgus macaques
clinical support
All animals received clinical support in the form of antibiotics and fresh irradiated
whole blood, as required. An prophylactic antibiotic regimen was initiated when
leukocyte count fell below 1,000/ µ l and continued daily until it exceeded 1,000/
µ l for three consecutive days: 1 ml/10 kg/day Bi-Gental® (Schering-Plough Santé
Animale) and 1 ml/10 kg Terramycin® (Pfizer). Fresh, irradiated (25 Gy; 137Cs
gamma radiation) whole blood (approximately 50 ml/transfusion) from a random
donor pool was administered if platelet count fell below 20,000/ µ l and hemoglo-
bin concentration was less than 6 g/dl.
Flow cytometry Analysis
Peripheral blood and bone marrow mononuclear cells were incubated for 30 min
at 4°C with 10 µ l of selected monoclonal antibodies for single- or triple-color
membrane staining. The following antibodies were used: APC-conjugated an-
ti-CD3 (SP34-2, Becton Dickinson), PE-conjugated anti-CD14 (clone M5E2,
BD Pharmingen), PE-conjugated anti-CD11b (BEAR-1, Beckman Coulter),
PerCP-conjugated anti-CD20 (clone B9E9, Immunotech), PE-conjugated anti
CD8 (clone RPA-T8, Becton Dickinson) and PerCP-conjugated antiCD4 (clone
L200, BD Pharmingen). Cells were washed twice and fixed in CellFix solution
(Becton Dickinson, Erembodegem, Belgium) for 3 days before analysis on a Bec-
ton Dickinson FACS apparatus with CellQuest Software (Becton Dickinson).
eGFP fluorescence was detected in the isothiocyanate (FITC) channel. Negative
controls from normal macaques were run with every experimental sample and
were used to establish gates for eGFP quantification.
Polymerase Chain Reaction (PCR) Assays
Cellular DNA was extracted from peripheral blood mononuclear cell (PBMC)
samples, using the High Pure PCR Template Preparation Kit according to the
manufacturer's instructions (Roche Mannheim, Germany). DNA was quantified
by measuring optical density (Spectra Max 190; Molecular Devices, California,
USA). The eGFP sequence was analyzed by quantitative real-time PCR on 250
ng of DNA run on an iCycler real-time thermocycler (Bio-Rad, California, USA).