Transgenic Blimp-1 Promoter Directs Hematopoietic Specific Gene Expression in Mice.

Abstract

B lymphocyte induced maturation protein-1 (Blimp-1) is a transcriptional repressor involved in B-lymphocyte differentiation and reported to be relatively plasma cell specific. The mechanisms that control its gene expression remain unknown although internal regulatory sequences have been postulated as required for B cell specificity. Aimed at directing therapeutic proteins into myeloma cells, we employed a transgenic strategy in studying the Blimp-1 promoter. We first cloned 5kb, 2.5kb and 1.6kb Blimp-1 promoter fragments from a BAC clone that contains the entire mouse Blimp-1 gene and fused the promoters to the luciferase reporter gene. As compared to a basal promoter PGL3, the Blimp-1 promoter fragments all demonstrated high and similar transcriptional activity (>7 fold over baseline) when transfected into BHK, Raji, OCIMy5 cells and R1 ES cells but weak activity (<2 fold) in murine preB leukemia cells. To further dissect Blimp-1 promoter function in vivo, the 5 kb promoter fragment (−4700 to +72bp) was fused to EGFP and used to generate transgenic mice. Northern blot and RT-PCR demonstrated that EGFP was expressed in the spleen, bone marrow (BM) and peripheral blood (PB) lymphocytes but not in the other tissue types examined. By FACS analysis, however, the EGFP positive population proved to be low (0.5–1%) in the spleen, PB or BM. To achieve higher Blimp-1 promoter-driven EGFP expression, we electroporated the 5kb Blimp-1/EGFP construct into ES cells and screened for higher EGFP expression clones. One clone with strong EGFP expression was used in blastocyst injection and generating chimeric mice. In this way, we have generated transgenic lines with higher EGFP expression in the spleen, BM and PB (3.2–4.7%, 2.2-3.4% and 3.8- 7.5%, respectively). After stimulation of cultured spleen and BM cells with 20 ug/ml LPS for 72 hrs, the EGFP population increased 2–3 fold in spleen cells and 3–4 fold in BM cells. Further FACS analyses of EGFP expression in LPS stimulated spleen cells demonstrated that EGFP was expressed in 14% of plasma cells (CD138+), 6.7% of mature B cells (CD19+), 4.6% of pre-B and B-cells (B220+) and 9.3% of CD8+ T cells. In BM culture after LPS plus IL-3 and IL-7 stimulation for 72 hr., EGFP is expressed in 8.7% of CD138+ cells, 5.9% of CD19+ cells, 6.9% of B220+ cells, 10% of TER119+ cells (erythropoietic cells), 11.3% of Sca-1+ cells and 12.8% cKit+ cells (progenitor cells). Furthermore, intravenous delivery of LPS resulted in upregulated EGFP expression in B cells, T cells and macrophages. The number of circulating B220-positive and EGFP-positive B cells doubled within 48hrs after LPS i.v. injection. Given EGFP expression in ES cells as well as Sca-1 and c-Kit+ve cells we examined emryos by fluorescent microscopy and observed strong EGFP expression in 9.5 dpc to 19.5 dpc mouse embryos. The precise localization of expression is now being analyzed. In summary: (1). The 5kb, 2.5kb and 1.6 kb Blimp-1 promoter fragments have similar transcriptional activity; (2). The 5kb Blimp-1 promoter is functional in vivo and specifically directs gene expression in all hematopoietic lineages. (3). The 5kb promoter is sufficient for LPS regulation. (4). The 5kb promoter is active in mouse embryonic development. As B cells were not specifically targeted we conclude that Blimp-1 has wider expression potential than previously recognized or that elements which control B cell specificity lie outside of the promoter fragments studied.