Isolation of stem cell-specific cDNAs from hematopoietic stem cell populations

被引：5

作者：

Orlic, D ^{[1
]}

Laprise, SL ^{[1
]}

Cline, AP ^{[1
]}

Anderson, SM ^{[1
]}

Bodine, DM ^{[1
]}

机构：

[1] NIH, Hematopoiesis Sect, Genet & Mol Biol Branch, Natl Human Genome Res Inst, Bethesda, MD 20892 USA

来源：

HEMATOPOIETIC STEM CELLS: BIOLOGY AND TRANSPLANTATION | 1999年 / 872卷

关键词：

D O I：

10.1111/j.1749-6632.1999.tb08469.x

中图分类号：

Q2 [细胞生物学];

学科分类号：

071009 ; 090102 ;

摘要：

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-kit(HI)) or depleted (Lin(-) c.kit(NEG)) 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.

引用

页码：243 / 255

页数：13

共 32 条

[1] Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries [J].