Development of a murine hematopoietic progenitor complementary DNA microarray using a subtracted complementary DNA library.
With the goal of creating a resource for in-depth study of myelopoiesis, we have executed a 2-pronged strategy to obtain a complementary DNA (cDNA) clone set enriched in hematopoietic genes. One aspect is a library subtraction to enrich for underrepresented transcripts present at early stages of hematopoiesis. For this, a hematopoietic cDNA library from primary murine bone marrow cells enriched for primitive progenitors was used as tester. The subtraction used 10 000 known genes and expressed sequence tags (ESTs) as driver. The 2304 randomly picked clones from the subtracted cDNA libraries represent 1255 distinct genes, of which 622 (50%) are named genes, 386 (30%) match uncharacterized ESTs, and 247 (20%) are novel. The second aspect of our strategy was to complement this subtracted library with genes known to be involved in myeloid cell differentiation and function. The resulting cDNAs were arrayed on polylysine-coated glass slides. The microarrays were used to analyze gene expression in primary and cultured murine bone marrow-derived progenitors. We found expression of various types of genes, including regulatory cytokines and their receptors, signal transduction genes, and transcription factors. To assess gene expression during myeloid differentiation, we examined patterns of change during induced differentiation of EML cells. Several hundred of the genes underwent fluctuations in expression level during myeloid cell differentiation. The complete database, accessible on the World Wide Web at http://yale130132115135.med.yale.edu/, allows for retrieval of information regarding these genes. Our microarray allows for genomewide expression analysis of myeloid stem cells, which will help in defining the regulatory mechanisms of stem cell differentiation.
Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06520-8023, USA.
Xianyong
X
Ma
Tupur
T
Husain
Hui
H
Peng
Sharon
S
Lin
Olga
O
Mironenko
Noel
N
Maun
Steven
S
Johnson
David
D
Tuck
Nancy
N
Berliner
Diane S
DS
Krause
Archibald S
AS
Perkins
D000818:Animals; D001854:Bone Marrow Cells; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling / Q000379:methods; D015723:Gene Library; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D019891:Leukopoiesis / Q000235:genetics*; D051379:Mice; D023461:Myeloid Progenitor Cells; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D013382:Subtraction Technique