American Society of Hematology

Fig. 5.

$Fig. 5. The 91-kb murine PU.1 genomic clone directs high-level reporter gene expression in myeloid and B cells of transgenic mice. / (A) Northern blot analysis demonstrates that the transgene is specifically expressed in the same tissues as the endogenous murine PU.1 gene. Total RNA was isolated from the different tissues as indicated at the top from either a nontransgenic littermate (Tg −) or a murine PU.1 91- kb P1 transgenic mouse (Tg +). The blot was successively hybridized with a probe for murine Thy1 (top), murine PU.1 (middle), and GAPDH (bottom). The Thy1 probe hybridized to transgene mRNA strongly in peritoneal macrophages (MΦ), bone marrow (BM), and spleen and weakly in liver and lung. The hybridization observed in both nontransgenic and transgenic thymus is derived from the endogenous murine Thy1 gene, which is expressed strongly in thymus, weakly in spleen and heart, and undetectable in the other tissues examined and indicated by an arrow on the right of the panel. Similar results were obtained with a second transgenic founder line. (B) Northern blot analysis of fractionated cells demonstrate myeloid- and B cell–specific expression of the PU.1 transgene. Cells from the P1 transgenic line shown in panel A were fractionated into B220+ (B cells); Gr-1+ (granulocytes); Mac-1+ (granulocytes and macrophages); and CD4+and/or CD8+ (T-cell) populations as described in “Materials and methods.” Murine Thy1.1, PU.1, and GAPDH RNAs were detected by Northern blot analysis as described for panel A. The endogenous murine Thy1 mRNA species was detected in the CD4+/CD8+ lane slightly below the position of the second fastest migrating exogenous transgene band, as indicated by the arrow to the right of the panel. The purity of cells for each lineage was more than 95% for Gr-1+ cells and more than 90% for Mac-1+, B220+, and CD4+/CD8+ cells. (C) RT-PCR analysis demonstrates expression of transgenic RNA in Mac-1+ myeloid cells but not TER119+ erythroid cells. Highly purified (> 99%) Mac-1+ (lane 1) and TER119+ cells (lane 2) were isolated by FACS. Complementary DNA from 200 cells was used in each RT-PCR reaction. Transgene-derived mRNA was detected by a combination of 5′ PU.1 sense and 3′ Thy1.1 antisense primers. GATA-1 and GAPDH RT-PCR were performed as controls for RNA integrity.$

The 91-kb murine PU.1 genomic clone directs high-level reporter gene expression in myeloid and B cells of transgenic mice.

(A) Northern blot analysis demonstrates that the transgene is specifically expressed in the same tissues as the endogenous murine PU.1 gene. Total RNA was isolated from the different tissues as indicated at the top from either a nontransgenic littermate (Tg −) or a murine PU.1 91- kb P1 transgenic mouse (Tg +). The blot was successively hybridized with a probe for murine Thy1 (top), murine PU.1 (middle), and GAPDH (bottom). The Thy1 probe hybridized to transgene mRNA strongly in peritoneal macrophages (MΦ), bone marrow (BM), and spleen and weakly in liver and lung. The hybridization observed in both nontransgenic and transgenic thymus is derived from the endogenous murine Thy1 gene, which is expressed strongly in thymus, weakly in spleen and heart, and undetectable in the other tissues examined and indicated by an arrow on the right of the panel. Similar results were obtained with a second transgenic founder line. (B) Northern blot analysis of fractionated cells demonstrate myeloid- and B cell–specific expression of the PU.1 transgene. Cells from the P1 transgenic line shown in panel A were fractionated into B220⁺ (B cells); Gr-1⁺ (granulocytes); Mac-1⁺ (granulocytes and macrophages); and CD4⁺and/or CD8⁺ (T-cell) populations as described in “Materials and methods.” Murine Thy1.1, PU.1, and GAPDH RNAs were detected by Northern blot analysis as described for panel A. The endogenous murine Thy1 mRNA species was detected in the CD4⁺/CD8⁺ lane slightly below the position of the second fastest migrating exogenous transgene band, as indicated by the arrow to the right of the panel. The purity of cells for each lineage was more than 95% for Gr-1⁺ cells and more than 90% for Mac-1⁺, B220⁺, and CD4⁺/CD8⁺ cells. (C) RT-PCR analysis demonstrates expression of transgenic RNA in Mac-1⁺ myeloid cells but not TER119⁺ erythroid cells. Highly purified (> 99%) Mac-1⁺ (lane 1) and TER119⁺ cells (lane 2) were isolated by FACS. Complementary DNA from 200 cells was used in each RT-PCR reaction. Transgene-derived mRNA was detected by a combination of 5′ PU.1 sense and 3′ Thy1.1 antisense primers. GATA-1 and GAPDH RT-PCR were performed as controls for RNA integrity.

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