Autologous Bone Marrow Transplantation - Blog Science Connections

7/2 Human ADA in Monkeys
supernatant. This "supernatant" procedure permitted us to treat a much
larger quantity of bone marrow cells and also resulted in much better recoveries
of treated cells after infection. Using this protocol, the animals' bone
marrow was rapidly reconstituted (see animals 5-7 and 9-12, Table 1).
Animals with fully reconstituted marrows were analyzed by Southern
blotting (8) to look for the presence of vector sequences integrated into the
genome, by neomycin phosphotransferase (NPT) assays (9) for the presence
of the bacterial neo R
marker gene, and by assays for human ADA gene
expression. Human and endogenous primate ADAs were separated by ionexchange
chromatography, after which the ADA activity in the human and
monkey regions was quantitated by thin-layer chromatography to measure
conversion of radioactive substrate (Fig 2) (10).
Overall, of the 12 animals that underwent bone marrow transplant/gene
transfer (Table 1), 8 could be analyzed as described above, but the marrows of
FRACTION NUMBER
Figure 2. Adenosine deaminase (ADA) and neomycin phosphotransferase (Neo R )
activity in primate bone marrow cells 10 weeks after transplantation—assay of monkey
no. 6 versus untreated control. The graph represents the 280-nm absorbance of bone
marrow lysate fractionated on a Pharmacia Fast Protein Liquid Chromatography Mono
Q column using a KCI salt gradient. Stippled bars represent ADA activity expressed as
the percent conversion of adenosine to inosine (%A~I). Fractions containing human
ADA, monkey ADA, and A/eo" enzyme activity are denoted.