The beta-galactosidase gene as a marker for hematopoietic reconstitution: individual cell analysis in a murine bone marrow transplantation model.
We have used a simple, single-gene retrovirus carrying the Escherichia coli beta-galactosidase reporter gene (lacZ), termed LlacZ. This virus was found to infect immortalized myeloid and lymphoid precursor/leukemic cell lines efficiently as well as primary murine bone marrow clonogenic progenitors, without apparent modulation of growth or phenotype. Following infection of bone marrow cells, a significant proportion of progenitors--36% of lineage-negative cells with low levels of c-kit expression (lin-/c-kit(lo)) known to be enriched with pluripotent hemopoietic stem cells, and 19% of Sca1-positive cells known to be enriched with transplantable cells with lymphomyeloid-reconstituting ability--were shown to express lacZ. Use of an LlacZ-infected population of post 5-fluorouracil bone marrow cells to reconstitute lethally irradiated mice demonstrated the presence of lacZ-expressing cells in the spleen at day 12 post-transplantation with provirus detected in individual spleen colonies (CFU-S). In the long term (3-6 months following transplantation), lacZ expression was detected in hematopoietic tissues of all recipient mice. The use of two-color in situ and flow cytometry analysis combined with lineage-specific antibodies showed lacZ expression in both myeloid and lymphoid cells in spleen and bone marrow. In addition, lacZ-expressing cells were detected in secondary recipient mice injected with bone marrow cells derived from primary LlacZ recipients. Overall, these data show the efficacy of a single gene vector for stem cell transduction, the utility of beta-galactosidase as a single cell marker for stem cell transduction and reconstitution ability, and the need for protocol optimization to see high-level multilineage gene expression.
Dolnikov
A
A
The School of Physiology and Pharmacology, The University of New South Wales, Randwick, NSW, Australia.
Millington
M
M
Beruter
E
E
Shounan
Y
Y
Symonds
G
G
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects / Q000528:radiation effects; D016026:Bone Marrow Transplantation; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005472:Fluorouracil / Q000633:toxicity; D005819:Genetic Markers; D006410:Hematopoiesis; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D023421:Models, Animal; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis / Q000235:genetics; D012190:Retroviridae; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics*
D005819:Genetic Markers; D019009:Proto-Oncogene Proteins c-kit; D001616:beta-Galactosidase; D005472:Fluorouracil