The numbers of lymphoid pre-B CFCs are severely reduced in
Hoxa9 transgenic mice. (A) Diagrammatic representation of the integrated Hoxa9 containing pLIT3 vector. This vector contains the full-length Hoxa9 cDNA, whose transcription is driven by the TCRVβgene, coupled to the μIgH chain enhancer and a fragment of the lck gene proximal promoter.19Sequences from the hGH gene, including splice sites and polyadenylation signal, are downstream of the Hoxa9 cDNA and are included in the primary transcript. (B) Total cellular RNA (10 μg) from the indicated tissues of homozygous mice from the 2Hoxa9 transgenic lines (12 and 15) and control littermates were analyzed by northern blot for the expression of theHoxa9 transgene using the full-length Hoxa9 cDNA as a probe. The expected 2.5 kb transgene transcript is detected mainly in bone marrow (B), spleen (S), and thymus (T), with low expression in lung (L), but is nondetectable in the kidney (K), heart (H), and brain (Br) tissues. A smaller transcript of 0.2 kb was detected in the spleen of both transgenic Hoxa9 lines. (C) Western blot analyses of total cell extracts from spleen (S) and thymus (T) obtained from transgenic Hoxa9 and control mice. Hoxa9 (upper panel) and PTP1D (loading control, bottom panel) levels are shown. (D) Evaluation of the numbers of lymphoid pre-B CFCs, WW-ICs, and myeloid CFCs in bone marrow of homozygous Hoxa9 transgenic mice. Results (mean ± SD) indicate percentages compared with controls (set at 100%) for 3 Hoxa9-12 and 3 Hoxa9-15 transgenic mice. (E) WW-IC–derived B220+ cells (at limiting dilution) were counted at day 21 of culture and are not statistically different (P < .13, 1-tailed Student t test with unequal variance, n = 4 mice in each group) between transgenics and controls. (F) Flow cytometric analysis of bone marrow, spleen, and thymic cells from the homozygous Hoxa9 transgenic mice.
