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Clock gene expression in purified mouse hematopoietic stem cells.
Oleg Tsinkalovsky1, Benedikte Rosenlund, Ole Didrik Laerum
1Stem Cell Research Group, Department of Pathology, the Gade Institute, Haukeland University Hospital, Bergen, Norway. oleg.tsinkalovsky@gades.uib.no
Experimental Hematology
|January 22, 2005
Summary
Researchers analyzed clock gene expression in mouse hematopoietic stem cells. High-speed flow sorting and quantitative PCR reliably measured gene activity in these rare cells.
Area of Science:
- Hematology
- Molecular Biology
- Chronobiology
Background:
- Circadian genes regulate biological rhythms but their role in hematopoietic stem cells (HSCs) remains uncharacterized.
- HSCs are rare in bone marrow, posing challenges for molecular analysis due to low cell yield and RNA quality concerns.
Purpose of the Study:
- To develop and validate a reliable method for analyzing clock gene expression in purified mouse HSCs.
- To overcome technical limitations in studying gene expression in small, rare cell populations.
Main Methods:
- Hematopoietic stem cells were enriched using high-speed flow cytometric cell sorting of side population (SP) cells from mouse bone marrow.
- Relative mRNA levels of six key clock genes (mPer1, mPer2, mBmal1, mCry1, mClock, mRev-erb alpha) were quantified using real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR).
- Cell clonogenicity was assessed via colony-formation assays to ensure the sorting method did not impair stem cell function.
Main Results:
- Significant differences in clock gene expression (mPer1, mCry1) were observed between sorted SP cells and whole bone marrow.
- High-speed sorting did not negatively impact clock gene expression or the clonogenicity of HSCs.
- The methodology proved robust even with a 24-hour delay in processing.
Conclusions:
- This study demonstrates the expression of six core clock genes within mouse hematopoietic stem cells.
- The combined approach of high-speed flow sorting and Q-RT-PCR is effective and reliable for analyzing clock gene activity in limited stem cell samples.