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PMID: 9611252 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

Nucleic acids research ·Vol. 26 ·No. 12 ·1998-06-15 ·Pages 3034-43

Iwama A, Zhang P, Darlington GJ, McKercher SR, Maki R, Tenen DG

Abstract

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.

MeSH Terms
Animals CCAAT-Enhancer-Binding Proteins Cell Line, Transformed Cloning, Molecular/methods Cytoplasmic Granules/genetics DNA-Binding Proteins/genetics,physiology Fetus Gene Expression Regulation, Developmental/physiology Genes/genetics Leukopoiesis/genetics Liver Mice Mice, Knockout Molecular Sequence Data Nuclear Proteins/genetics,physiology Nucleic Acid Hybridization Polymerase Chain Reaction/methods Proto-Oncogene Proteins/genetics,physiology RNA, Messenger/analysis Receptors, Colony-Stimulating Factor/genetics Trans-Activators/genetics,physiology Transcription Factors/physiology
Chemicals
CCAAT-Enhancer-Binding Proteins DNA-Binding Proteins Nuclear Proteins Proto-Oncogene Proteins RNA, Messenger Receptors, Colony-Stimulating Factor Trans-Activators Transcription Factors proto-oncogene protein Spi-1
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Iwama A
Hematology/Oncology Division and Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA.
Zhang P
Darlington G J
McKercher S R
Maki R
Tenen D G
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/26.12.3034
Published
1998-06-15
Pages
3034-43
Language
English
Region
England
NLM ID
0411011
PMCID
PMC147647
Subset
IM
Grants
NIAID NIH HHS · AI30656 · United States
NCI NIH HHS · CA41456 · United States
NIDDK NIH HHS · DK48660 · United States
Databases
GENBANK
AA720492, AA720493, AA720494, AA720495, AA720496, AA720497, AA720498, AA720499, AA720500, AA720501
External Links
PubMed source DOI: 10.1093/nar/26.12.3034 PMC Full Text View journal details
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