サイトメトリーの発生生物学・再生医学への応用 緑色蛍光蛋白質を用いた造血組織発生における転写因子GATA-2の発現と機能の解析

抄録

The green fluorescent protein (GFP) produces the fluorescence similar to that of fluorescein isothiocyanate,within living cells. Using flowcytometry, we can analyze and sort the cells expressing GFP fluorescence to accomplish the cellular and molecular biological experiments. We made the transgenic mice, and the knockin mice, in which the expression of the GFP protein was driven by the transcriptional activity of the GATA-2 gene regulatory region. The GATA-2 is a transcription factor essential to the hematopoiesis, but its roles are not clarified yet. These mice produced the fluorescence in the hematopoietic cells, recapitulating the endogenous expression of the GATA-2 gene. The GFP expressing cells in the fetal liver had the same surface marker profile to that of hematopoietic stem/progenitor cells. We found that the GFP was also expressed in the aorta and adjacent tissues, where the production of adult-type hematopoietic stem/progenitor cells occurred. These cells had the morphology of the mesenchymal cells and expressed no CD45 hematopoietic cell antigens, but produced abundant hematopoietic cells after culture, indicating that the GATA-2 was expressed in the early precursors of the hematopoietic cells. Further, we used the GFP to identify the cells infected by retrovirus, and revealed the inhibitory role of GATA-2 expression to produce hematopoietic cells from these precursor mesenchymal cells. Thus, the GFP techniques with flowcytometry, combined with the developmental biotechnology, are now the strong tool to reveal the physiological molecular mechanisms in the individual cells forming mammalian tissues.