Isolation of stem cell-specific cDNAs from hematopoietic stem cell populations.
We have begun to isolate gene sequences that are specifically expressed in hematopoietic stem cells (HSCs). There are at least three fundamental requirements for the isolation of HSC-specific transcripts. First, highly enriched populations of HSCs, and an HSC-depleted cell population for comparison must be isolated. Secondly, the gene isolation procedures must be adapted to accommodate the small amounts of RNA obtained from purified HSCs. Finally, a defined screening strategy must be developed to focus on sequences to be examined in more detail. In this report, we describe the characterization of populations of HSCs that are highly enriched (Lin- c-kitHI) or depleted (Lin- c-kitNEG) of HSCs. We compared two methods for gene isolation, differential display polymerase chain reaction (DD-PCR) and subtractive hybridization (SH), and found that the latter was more powerful and efficient in our hands. Lastly we describe the strategy that we have developed to screen clones for further study.
Orlic
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Hematopoiesis Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892-4442, USA.
Laprise
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SL
Cline
A P
AP
Anderson
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SM
Bodine
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DM
D000740:Anemia / Q000235:genetics; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002469:Cell Separation / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D015723:Gene Library; D005914:Globins / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D016133:Polymerase Chain Reaction; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis / Q000235:genetics; D014158:Transcription, Genetic; D001613:beta 2-Microglobulin / Q000235:genetics
D018076:DNA, Complementary; D001613:beta 2-Microglobulin; D005914:Globins; D019009:Proto-Oncogene Proteins c-kit