Use of RDA analysis of knockout mice to identify myeloid genes regulated in vivo by PU.1 and C/EBPalpha.
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, we studied the role of PU.1 and C/EBPalpha in the regulation of myeloid target genes in vivo . Northern blot analysis revealed that mRNAs encoding receptors for 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, we performed representational difference analysis (RDA), a PCR-based subtractive hybridization using fetal livers from wild type and PU.1 or C/EBPalpha knockout mice. By introducing a new modification of RDA, that of tissue-specific gene suppression, we could selectively identify a set of differentially expressed genes specific to myeloid cells. Differentially expressed genes included both primary and secondary granule protein genes. In addition, eight novel genes were identified that were upregulated in expression during myeloid differentiation. These methods provide a general strategy for elucidating the genes affected in murine knockout models.
Iwama
A
A
Hematology/Oncology Division and Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
Zhang
P
P
Darlington
G J
GJ
McKercher
S R
SR
Maki
R
R
Tenen
D G
DG
D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D002461:Cell Line, Transformed; D003001:Cloning, Molecular / Q000379:methods; D003594:Cytoplasmic Granules / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D005333:Fetus; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D005796:Genes / Q000235:genetics; D019891:Leukopoiesis / Q000235:genetics; D008099:Liver; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D016184:Receptors, Colony-Stimulating Factor / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D000099232:Proto-Oncogene Protein Spi-1
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016184:Receptors, Colony-Stimulating Factor; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1