Assessment of transduction rates of porcine bone marrow

Although drug resistance is commonly used as an indicator of gene transfer in various cellular contexts, the assessment of drug resistance is often im precise and over-estimated. To measure accurately transduction efficiencies of the retroviral-mediated transfer of genes encoding the neomycine phosph otransferase (Neo(r)) and porcine major histocompatibility (MHC) class II i n pig bone marrow cells (BMC), the fraction of targeted progenitors was eva luated by both colony-forming unit granulocytes/macrophages assays (G418(r) CFU-GM) and by PCR analysis of the transgenes (Tg). Transduced and untrans duced BMC were selected at different concentrations of G418 and revealed hi gh individual variability of drug sensitivity. Comparison of the results ob tained by estimating the CFU frequency and the PCR assays on drug-resistant colonies demonstrated a marked overestimation of BM transduction rates whe n determined by G418 resistance alone, because only approximately one-third of individual colonies were positive for both the Neo(r) and the class II Tg. Because this discrepancy is likely to affect the overall assessment of transduction rates using drug resistance markers, our data attest for the n eed of a combination of molecular assays to determine transduction efficien cies accurately.