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 130

days on semi-solid medium, as described above, to allow their myeloid differentia-
tion into more mature cells.
AZT Pretreatment of Immunoselected CD34 + cells
CD34 + cells were treated with AZT before transduction, to inhibit transduction
due to reverse transcription of the lentiviral vector genome. Immunoselected
CD34+ cells were cultured overnight in the proliferation medium described above,
with AZT concentrations of 0, 10 -7 , 10 -6 and 10 -5 molar. The cells were washed
twice and transduced with the lentiviral vector, according to the protocol described
above. The real percentage of GFP-positive cells resulting from reverse transcrip-
tion of the lentiviral vector was thus determined by subtracting the percentage of
GFP-positive cells obtained after treatment with a saturating dose of AZT, from
the percentage of GFP-positive cells obtained in the absence of AZT treatment.
Fluorescence Microscopy
After transduction and myeloid differentiation in semi-solid medium, the colonies
formed by AZT-treated CFC were observed by fluorescence microscopy (Axiovert
S100, Zeiss) using a magnification factor of 100. Fluorescence microscopy was
used to detect GFP in each colony subtype, making it possible to determine the
percentage of the colonies positive for GFP. We considered all colonies containing
GFP-producing cells to be GFP-positive. Images were analyzed with Adobe Pre-
miere and Adobe Photoshop software (Adobe Systems Inc., San Jose, CA, USA).
Gamma irradiation
Eight animals were sedated with ketamine (Imalgène; 10 mg/kg, i.m., Rhône-
Mérieux, France) and placed in a restraint chair. They received myeloablative
conditioning, in the form of total body exposure to 60Co gamma rays with an
anterior unilateral direction. A total midline tissue dose of 6 Gy was delivered at
a rate of 25.92 cGy/minute. Dosimetry was performed, with 100 µ L ionization
chambers placed in paraffin wax cylindrical phantoms of a similar size and orien-
tation to the seated animal.
Transplantation of Modified CD34 + bone Marrow cells
After the coculture of CD34 + cells with the lentiviral vector, four animals under-
went intramedullary infusion, of whole immunoselected CD34 + cells into both
humeri (Table 1).