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Selective rescue of early haematopoietic progenitors in Scl^–/– mice by expressing Scl under the control of a stem cell enhancer

¹ University of Cambridge, Department of Haematology, CIMR Centre, Hills Road, Cambridge CB2 2XY, UK
² Johannes Gutenberg-University, Institute of Toxicology, Laboratory of Molecular Mouse Genetics, Obere Zahlbacher Str. 67, 55131 Mainz, Germany

View larger version (37K): [in a new window]   Fig. 1. The Scl 3' enhancer construct targets lacZ expression in transgenic mice to long-term reconstituting haematopoietic stem cells from adult bone marrow and foetal liver. (A) FACS analysis of cell suspensions from non transgenic and transgenic adult bone marrow for the expression of lacZ by FDG staining on gated live cells (PI-negative cells). Histograms for FDG expression in one representative experiment are shown. Cells included in the sorted windows were designated as FDG– and FDG+. (B) Ethidium bromide-stained gels of peripheral blood PCR analysis for the presence of the transgene (lacZ) together with an internal control (myogenin). PCR products are shown from a representative experiment performed 5 months post-transplantation with bone marrow cells. Lanes correspond to individual animals transplanted with the indicated number of FDG+ or FDG– cells. Peripheral blood from transgenic and non-transgenic mice was mixed to provide 100%, 10%, 1% and 0% lacZ controls, allowing semi-quantitative evaluation of reconstitution levels. (C) Multilineage analysis in one recipient mouse 12 months post-transplant with FDG+ bone marrow cells. (D) FACS analysis of cell suspensions from non transgenic and transgenic foetal liver for the expression of lacZ by FDG staining on gated live cells (PI-negative cells). Histograms for FDG expression in one representative experiment from E12 liver cells are shown. (E) lacZ-PCR analysis of peripheral blood 4 months post-transplantation with foetal liver cells. (F) Multilineage analysis of a recipient mouse 6 months post transplant with foetal liver FDG+ cells. Bb, B lymphocytes B220+ from bone marrow; Bs, B lymphocytes B220+ from spleen; Bl, blood; BM, bone marrow; Erb, erythocytes Ter119+ from bone marrow; Grb, granulocytes Gr-1+ from bone marrow; LN, lymph nodes; Mø*, peritoneal cells; Møb, macrophages CD11b+ from bone marrow; Møs, macrophages CD11b+ from spleen; Sp, spleen; Th, thymus; Ts, T lymphocytes CD4+CD8+ from spleen; TFL, total foetal liver. The 2262 transgenic line was used in the experiments shown.

View larger version (76K): [in a new window]   Fig. 2. The Scl 3' enhancer construct targets lacZ expression to c-Kit+CD34+ cells from the AGM region. Cell suspensions from the AGM region of +6E5/lacZ/3'En transgenic E11 embryos were stained with biotinyl-conjugated anti-CD34 and PE-conjugated anti-c-Kit antibodies followed by streptavidin-FITC. Cells were sorted according to indicated windows. After sorting, cytospin preparations were stained for ß-galactosidase. In the representative experiment shown, approximately 70% of the c-Kit+CD34+ cells were lacZ+, compared with fewer than 1% of the c-Kit+CD34– population. Dead cells were excluded by PI staining.

View larger version (56K): [in a new window]   Fig. 3. Expression of exogenous Scl under control of the Scl 3' enhancer rescues haematopoiesis in Scl–/– embryos. (A) The +6E5/Scl-lacZ/3'En transgene. (B) X-gal staining of E7 embryo carrying the +6E5/Scl-lacZ/3'En transgene showing lacZ expression in the extra-embryonic mesoderm. (C-F) E8 embryos of the indicated genotype analysed by whole-mount (C, E) or cryostat sections (D,F) for lacZ staining. Note the presence of small numbers of round haematopoietic cells in blood islands of rescued yolk sacs (F). h, heart; p-sp, para-aortic splanchnopleura; ys, yolk sac; arrows, lacZ+ blood cells; arrowheads, lacZ+ endothelial cells. (G-O) E9 embryos of the indicated genotypes. Unfixed Tg+/–Scl–/– embryos were anaemic (compare G with J and M). Histological sections of wax-embedded X-gal-stained embryos (H,I,K,L,N,O) demonstrated the presence of haematopoietic cells in rescued embryos (K,L). Embryos are from transgenic line 2708 and were genotyped by PCR.

View larger version (61K): [in a new window]   Fig. 4. Myeloid and erythroid colony forming cells are obtained from cultures of Tg+/–Scl–/– yolk sacs. (A) FACS analysis of yolk sacs from E9 embryos. Cell suspensions from individual yolk sacs were stained with the indicated antibodies and body remnants used for lacZ/Scl PCR genotyping. Double staining was performed using FITC-conjugated c-Kit with PE-conjugated Ter119. Quadrants were established according to values obtained using cells stained with isotype controls in the same experiment. (B) Two erythroid colonies containing diaminofluorene positive haemoglobinised cells (arrows) from Tg+/–Scl–/– embryos are shown. (C) PCR analysis of individual colonies from yolk sacs of the indicated genotypes. (D) May-Grunwald-Giensa staining of cytospin preparation of yolk sac haematopoietic colonies obtained from embryos of the indicated genotype. Myeloid cells were seen in colonies from both Tg+/–Scl–/– and Tg+/–Scl+/– embryos. Normoblasts (arrows) were only seen in colonies from Tg+/–Scl+/–.

View larger version (89K): [in a new window]   Fig. 5. Anti-PECAM1 antibody staining of E9.5 embryos. Large branching vitelline vessels in the yolk sac (white arrows) are prominent in wild type Tg+/–Scl+/– embryos (A) as well as in rescued Tg+/–Scl–/– embryos (C) but were not observed in mutant Tg–/–Scl–/– yolk sacs (B). Inter-somitic vessels (arrowhead) are also well formed in both wild-type (D) and rescued embryos (F) but not in the mutant embryo (E). The head of the embryos were used to determine the embryo genotype.