USE OF RDA ANALYSIS OF KNOCKOUT MICE TO IDENTIFY MYELOID GENES REGULATED IN-VIVO BY PU.1 AND C EBP-ALPHA/

PU.1 and C/EBP alpha are transcription factors essential for normal my eloid development, Loss-of-function mutation of PU.1 leads to an absol ute block in monocyte/ macrophage development and abnormal granulocyti c development while that of C/EBP alpha causes a selective block in ne utrophilic differentiation. In order to understand these phenotypes, w e studied the role of PU.1 and c/EBP alpha in the regulation of myeloi d target genes in vivo. Northern blot analysis revealed that mRNAs enc oding receptors for M-CSF, G-CSF and GM-CSF, were expressed at low lev els in PU.1(-/-) fetal liver compared with wild type. To identify addi tional myeloid genes regulated by PU.1 and C/EBP alpha, we performed r epresentational difference analysis (RDA), a PCR-based subtractive hyb ridization using fetal livers from wild type and PU.1 or C/EBP alpha k nockout mice. By introducing a mew modification of RDA, that of tissue -specific gene suppression, we could selectively Identify a set of dif ferentially expressed genes specific to myeloid cells, Differentially expressed genes included both primary and secondary granule protein ge nes. In addition, eight novel genes were identified that were upregula ted in expression during myeloid differentiation, These methods provid e a general strategy for elucidating the genes affected in murine knoc kout models.