Use of RDA analysis of knockout mice to identify myeloid genes regulated in vivo by PU.1 and C/EBPalpha.

摘要

PU.1 and C/EBPalpha are transcription factors essential for normal myeloid development. Loss-of-function mutation of PU.1 leads to an absolute block in monocyte/macrophage development and abnormal granulocytic development while that of C/EBPalpha causes a selective block in neutrophilic differentiation. In order to understand these phenotypes, the role of PU.1 and C/EBPalpha in the regulation of myeloid target genes in vivo was studied. Northern blot analysis revealed that mRNAs encoding receptorsfor M-CSF, G-CSF and GM-CSF were expressed at low levels in PU.1-/- fetal liver compared with wild-type. To identify additional myeloid genes regulated by PU.1 and C/EBPalpha, representational difference analysis (RDA), a PCR-based subtractive hybridization, was performed using fetal livers from wild type and PU.1 or C/EBPalpha knockout mice. A new modification of RDA, that of tissue-specific gene suppression, permitted the selective identification of a set of differentially expressed genes specific tomyeloid cells. Differentially expressed genes included both primary and secondary granule protein genes. Eight novel genes were identified that were up-regulated in expression during myeloid differentiation.