Analysis of gene expression in small numbers of purified hemopoietic progenitor cells by RT-PCR.
Primitive hemopoietic stem cells represent the most probable targets for genetic alterations due to exposure to ionizing irradiation or chemical carcinogens. We have applied a two-step protocol for the purification of CD34+HLA-DR-/low hemopoietic progenitor cells from cord blood (CB). CD34+ cells were isolated by monoclonal antibody (mAb) against CD34 (My10) and immunomagnetic beads. Beads were cleaved off the CD34+ cells by enzymatic treatment with chymopapain. Due to chymopapain-resistance of epitopes recognized by the used mAbs purity control of CD34+ cells and separation into CD34+HLA-DR-/low and CD34+HLA-DR+ subsets could be performed by using flow cytometry. Two miniaturized procedures were applied to isolate poly(A)+ mRNA for the reverse transcription polymerase chain reaction (RT-PCR) from small numbers of CD34+HLA-DR-/low cells. In five experiments, the mean purity of immunomagnetically isolated CD34+ cells was 93.8% +/- 3.9. Flow cytometry sorting of CD34+ cells resulted in pure CD34+HLA-DR-/low populations (purity > 98.8%; range 98.8% to 99.9%; viability > 96%) with an average yield of 2600 +/- 800 cells/5 x 10(7) low density CB cells. By RT-PCR using both poly(A)+ mRNA isolation procedures, sequences corresponding to CD34 and beta 2-microglobulin were amplified from as few as 20 cells. Furthermore, a sequence-independent RT-PCR (SIP-RT-PCR) was applied to amplify the cDNA derived from five erythroblasts isolated from a burst-forming unit-erythroid (BFU-E). Upon hybridization, full-length c-fos message was detected in the SIP-RT-PCR amplified material. Our data demonstrate that gene expression can be detected at the transcriptional level in small numbers of hemopoietic progenitor cells. In addition, the SIP-RT-PCR may allow the amplification of unique mRNA species when subtractive hybridization procedures are performed. The presented data should be useful to analyze gene expression in rare subsets of radiation-exposed immature hemopoietic stem/progenitor cells.
D018952:Antigens, CD34 / Q000378:metabolism; D001483:Base Sequence; D002469:Cell Separation / Q000379:methods; D002917:Chymopapain; D017931:DNA Primers / Q000235:genetics; D005260:Female; D005312:Fetal Blood / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D006684:HLA-DR Antigens / Q000235:genetics / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D018952:Antigens, CD34; D017931:DNA Primers; D006684:HLA-DR Antigens; D012333:RNA, Messenger; D002917:Chymopapain