The ultimate goal of human gene therapy in treating hematopoietic disorders is to insert a functional copy of the affected gene into self-renewing stem cells. The engineered pluripotent cells should then provide all lineages of corrected blood cells for the lifetime of the recipient. It is therefore important to develop methods of tracking and studying the progeny of individual human hematopoietic stem cells. Using the technique of single-colony inverse polymerase chain reaction (PCR), we assessed the clonal diversity of marked colony-forming cells that had developed from transduced human hematopoietic progenitors in a long-term xenograft system. The LN retroviral vector, which carries the neo gene, was used to individually mark human CD34+ progenitors. The marked cells were then transplanted into immune-deficient mice for periods of up to 1 year to assess their survival and retention of clonogenic capacity. Following long-term engraftment, bone marrow cells recovered from each mouse were plated in human-specific colony-forming unit (CFU) assay with and without the drug G418, which selects for cells expressing the neo gene. Three weeks later, well-isolated colonies that had grown in G418 were plucked, and PCR for the neo gene was performed to confirm the presence of the vector. Inverse PCR was then performed on neo+ colonies to analyze the integration site of the LN provirus in human DNA. The clonal diversity of G418-resistant (G418R) human CFU recovered from 18 long-term engrafted beige/nude/xid (bnx) mice was assessed. From one to six human hematopoietic precursors had generated all marked colony-forming progenitors (3-39) recovered from the marrow of each animal. To assess the extent of in vitro self-renewal divisions, marrow samples from 22 sets of experiments, with 2-4 mice transplanted in each set, were studied using the single-colony inverse PCR technique. Proviral integrants at identical sites were found in only two mice transplanted with cells transduced in the same flask. The presence of identical integration sites in human progenitors recovered from two mice demonstrated that a long-lived, marked cell had self-renewed in vitro before transplantation and that both daughter cells had retained the capacity to home to the bnx bone marrow and survive for 10 months. Our in vivo xenograft model and the inverse PCR technique have allowed us to identify, trace, and quantitate the clonogenic progeny of primitive human hematopoietic cells for up to 1 year after retroviral-mediated transduction.