Characterization of hematopoietic progenitor cells that express the transcription factor SCL, using a lacZ "knock-in" strategy - PubMed (original) (raw)
Characterization of hematopoietic progenitor cells that express the transcription factor SCL, using a lacZ "knock-in" strategy
A G Elefanty et al. Proc Natl Acad Sci U S A. 1998.
Abstract
Gene targeting experiments have demonstrated that the transcription factor SCL is essential for primitive and definitive hematopoiesis in the mouse. To study the functional properties of hematopoietic cells expressing SCL, we have generated mutant mice (SCLlacZ/w) in which the Escherichia coli lacZ reporter gene has been "knocked in" to the SCL locus, thereby linking beta-galactosidase expression to transcription from the SCL promoter. Bone marrow cells from heterozygous SCLlacZ/w mice were sorted into fractions expressing high, intermediate and low levels of beta-galactosidase (designated lacZhigh, lacZint, and lacZneg). Cells that were lacZhigh or lacZint were enriched for day 12 spleen colony-forming units and myeloid and erythroid colony-forming cells (CFCs). These fractions included >99% of the erythroid and >90% of the myeloid CFCs. Culture of sorted bone marrow populations on stromal cells secreting interleukin-7 or in fetal thymic organ cultures showed that B and T lymphoid progenitors were also present in the lacZhigh and lacZint fractions. These data provide a functional correlation between SCL expression and colony-forming ability in immature hematopoietic cells. Our data also suggested that expression of SCL was transient and confined to hematopoietic stem and/or progenitor cells, because the differentiated progeny of most lineages (except the erythroid) were beta-galactosidase-negative.
Figures
Figure 1
SCL-lacZ gene targeting strategy. (A) The SCL-lacZ targeting vector is shown above the map of the SCL genomic locus. Regions of homology where recombination occurred are indicated as crosses and the correctly targeted locus is depicted below. Exons are shown as numbered, solid boxes. The positions of the E. coli lacZ_gene (lacZ), the polyadenylation signal (pA), and the_PGKneo cassette (neo) are indicated. Abbreviations: B,_Bam_HI; R, _Eco_RI; S, _Sac_I; X, _Xba_I. (B) Southern hybridization of_Bam_HI-digested DNA from wild-type (w/w) and heterozygous (lacZ/w) mice hybridized with probe A, indicating germ-line (5 kb) and targeted (3 kb) alleles.
Figure 2
Expression of β-galactosidase in_SCL_lacZ/w bone marrow cells. (A) Representative FACS profiles of forward scatter plotted against β-galactosidase activity (measured as fluorescence) for FACS-Gal-labeled bone marrow cells from_SCL_lacZ/w and_SCL_w/w mice. Gating windows and the percentage of cells in each window are shown for lacZneg, lacZint, and lacZhigh fractions. (B) May-Grünwald-Giemsa stained cytocentrifuge preparations of unsorted and sorted bone marrow fractions from_SCL_lacZ/w mice.
Figure 3
Enrichment for myeloid and erythroid CFCs in the lacZ-positive bone marrow fractions of_SCL_lacZ/w mice. (A) Frequency of day 7 myeloid CFC in agar cultures of unsorted and sorted bone marrow fractions from _SCL_lacZ/w and_SCL_w/w mice cultured in IL-3 or SCF. Values represent the mean ± SD from five experiments for_SCL_lacZ/w and the mean from two experiments for _SCL_w/w bone marrow cells. The percentage of bone marrow cells that were sorted into each fraction is indicated. (B) Frequency of day 2 CFU-E, day 7 BFU-E, and myeloid (GM) CFC in methylcellulose cultures of unsorted and sorted bone marrow fractions from _SCL_lacZ/w mice cultured in Epo (for day 2 CFU-E) or IL-3/Epo (for day 7 BFU-E and GM CFC). Values represent the mean ± SD from three experiments. The percentage of bone marrow cells that were sorted into each fraction is indicated.
Figure 4
B lymphoid progenitor cells are found in lacZ-positive and -negative bone marrow fractions of_SCL_lacZ/w mice. Flow cytometric profiles of cells harvested from IL-7/NIH 3T3 cultures seeded with unsorted or sorted bone marrow fractions and labeled with directly conjugated antibodies against the B lymphoid antigen, B220, and the myeloid antigen, Mac-1.
Figure 5
T lymphoid cell surface markers expressed by the progeny of bone marrow cells reconstituting fetal thymic lobes in organ culture. Identical flow cytometric profiles were obtained regardless of whether β-galactosidase-positive or -negative bone marrow fractions were used to repopulate the thymic lobes.
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