Clonal Marking of Mouse Hematopoietic Stem and Lymphoid Progenitor Cell Populations.

We have developed a method to clonally mark hematopoietic stem and lymphoid progenitor cell populations using a novel sequence tag approach. A library containing an 11-base random sequence tag is cloned into a lentivirus vector, packaged using the VSV-G glycoprotein and HIV-1 capsid, and transduced into freshly isolated mouse hematopoietic stem cell (Thy-1.1^lowc-kit^high) or progenitor cell (Thy-1.1^negc-kit^high) populations. To minimize artifacts introduced by prolonged culture, we have utilized a 3-hour spinoculation protocol performed in the absence of cytokines. Transduction efficiency was evaluated in vitro by methylcellulose colony assay and liquid cultures, and in vivo by transplanting the transduced cells into lethally irradiated mice. A bicistronic lentivirus vector with a CMV promoter driving expression of a transcript encoding Thy-1.2-IRES-GFP was used to optimize the transduction protocol. Liquid culture assays demonstrated 57% transduction efficiency after 5 days of growth, based on expression of the Thy-1.2 and GFP reporter proteins. Mice transplanted with transduced Thy-1.1^negc-kit^high progenitor cells were sacrificed after 16 days, a time at which we have previously observed robust progenitor cell engraftment in the thymus while progeny of Thy-1.1^lowc-kit^high HSC have not yet appeared. In 4 of 4 transplanted mice, we observed donor-derived cells in the bone marrow, lymph nodes and thymus. The percentage of total cells expressing the lentivirus-derived transgene ranged from 1.6% of bone marrow cells to 20% of thymocytes. Peripheral blood from mice transplanted with transduced HSC were analyzed and monitored every 4 weeks for transgene expression. We observed that although the Thy-1.2 marker was expressed and maintained up to 14 weeks after HSC transplant, GFP transgene expression was minimal. Based on these preliminary results, we have engineered a new lentivirus vector containing random sequence tags and the Thy-1.2 marker. This strategy provides a simple and efficient way of tracking the progeny of individual cells within a transplanted population, using PCR amplification of the random tags found within mature cell populations derived from the transduced cells. Sequence analysis of individual clones derived from different lineages of cells will enable us to better define the lineage potentials of specific progenitor cell subpopulations.