Transgenic Analysis of the Stem Cell Leukemia +19 Stem Cell Enhancer in Adult and Embryonic Hematopoietic and Endothelial Cells

Abstract

Appropriate transcriptional regulation is critical for the biological functions of many key regulatory genes, including the stem cell leukemia (SCL) gene. As part of a systematic dissection of SCL transcriptional regulation, we have previously identified a 5,245-bp SCL +18/19 enhancer that targeted embryonic endothelium together with embryonic and adult hematopoietic progenitors and stem cells (HSCs). This enhancer is proving to be a powerful tool for manipulating hematopoietic progenitors and stem cells, but the design and interpretation of such transgenic studies require a detailed understanding of enhancer activity in vivo. In this study, we demonstrate that the +18/19 enhancer is active in mast cells, megakaryocytes, and adult endothelium. A 644-bp +19 core enhancer exhibited similar temporal and spatial activity to the 5,245-bp +18/19 fragment both during development and in adult mice. Unlike the +18/19 enhancer, the +19 core enhancer was only active in adult mice when linked to the eukaryotic reporter gene human placental alkaline phosphatase. Activity of a single core enhancer in HSCs, endothelium, mast cells, and megakaryocytes suggests possible overlaps in their respective transcriptional programs. Moreover, activity in a proportion of thymocytes and other SCL-negative cell types suggests the existence of a silencer elsewhere in the SCL locus.

Organ-injury-induced reactivation of hemangioblastic precursor cells

“…30 Bone marrow cells were obtained from SCL-þ 19 PLAP transgenic mice and prepared as previously described. 23 Bone marrow cells (5 Â 10 6 cell) suspension is injected in 50 ml of D-PBS 1% fetal calf serum. When primary recipient animals have reached 1 month of age, blood is retrieved from the tail and subject to PLAP-PCR blood chimerism analysis.…”

“…For fluorescence-activated cell sorting (FACS) analysis, bone marrow cells from transgenic mice were stained according to the protocol described in the following section as previously described. 23 Cells were stained with the antibodies PLAP, Ter119PE, CD45PE and CD31bio followed by incubation with anti-rabbit-FITC and stretavidine-cy5.…”

“…Cells were analyzed by flow cytometry (FACS) using a FACSAria flow cytometer (BD Biosciences) according to the protocol previously described. 23 Peripheral blood chimerism was assessed by quantification of the percentage of PLAP þ cells compared to the levels in SV/PLAP/19 transgenic and nontransgenic mice. To confirm multilineage hematopoietic engraftment, animals with high levels of peripheral blood PLAP þ cells were killed and genomic DNA was isolated from spleen, lymph nodes, thymus and bone marrow or FACS-sorted cells and analyzed by PCR for the presence of the PLAP transgene as published.…”

“…To confirm multilineage hematopoietic engraftment, animals with high levels of peripheral blood PLAP þ cells were killed and genomic DNA was isolated from spleen, lymph nodes, thymus and bone marrow or FACS-sorted cells and analyzed by PCR for the presence of the PLAP transgene as published. 23 …”

“…In adult mice, the 3 0 En is active in the vast majority of hematopoietic stem cells and progenitors present in bone marrow 20 and recent evidence suggests also expression in some adult endothelial cells. 23 Unlike the þ 18/19 enhancer whose activity was assessed in transgenic mice carrying a LacZ/ þ 18/ 19 reporter construct, the þ 19 core enhancer was active only in adult mice when linked to the eukaryotic reporter gene human placental alkaline phosphatase (PLAP) 23 .…”