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A Genetic Strategy for Single and Combinatorial Analysis of miRNA Function in Mammalian Hematopoietic Stem Cells†‡§

  1. Eirini P. Papapetrou1,2,¶,*,
  2. James E. Korkola3,
  3. Michel Sadelain1,2

Article first published online: 12 NOV 2009

DOI: 10.1002/stem.257

Issue

STEM CELLS

STEM CELLS

Volume 28, Issue 2, pages 287–296, February 2010

Additional Information

How to Cite

Papapetrou, E. P., Korkola, J. E. and Sadelain, M. (2010), A Genetic Strategy for Single and Combinatorial Analysis of miRNA Function in Mammalian Hematopoietic Stem Cells. STEM CELLS, 28: 287–296. doi: 10.1002/stem.257

Author Information

  1. 1

    Center for Cell EngineeringMemorial Sloan-Kettering Cancer Center, New York, New York, USA

  2. 2

    Molecular Pharmacology and Chemistry ProgramMemorial Sloan-Kettering Cancer Center, New York, New York, USA

  3. 3

    Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

  1. Telephone: 212-639-6170, fax: 917-432-2340

*Center for Cell Engineering, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY, 10065, USA

  1. †

    Author contributions: E.P.P.: Conception and design, collection, analysis and interpretation of data, manuscript writing; J.E.K.: Collection and analysis of microarray data; M.S.: Design, analysis and interpretation of data, manuscript writing, financial support, final approval of manuscript.

  2. ‡

    Disclosure of potential conflicts of interest is found at the end of this article.

  3. §

    First published online in STEM CELLSEXPRESS January 28, 2010.

Publication History

  1. Issue published online: 16 FEB 2010
  2. Article first published online: 12 NOV 2009
  3. Accepted manuscript online: 12 NOV 2009 12:00AM EST
  4. Manuscript Accepted: 4 NOV 2009
  5. Manuscript Received: 30 JUN 2009

Funded by

  • NIH. Grant Numbers: HL 57612, PO1 CA059350
  • New York State Stem Cell Science. Grant Number: NYSTEM-016

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Keywords:

  • miRNA;
  • miR-144;
  • miR-451;
  • erythropoiesis;
  • lentiviral vectors;
  • hematopoietic stem cells

Abstract

The regulatory role of micro-RNAs (miRNAs) in hematopoietic development is increasingly appreciated. Reverse genetics strategies based on the targeted disruption of miRNAs offer a powerful tool to study miRNA functions in mammalian hematopoiesis. The miR-144/451 cluster comprises two miRNAs coexpressed from a common precursor transcript in an erythroid-specific manner. To decipher the contribution of each miRNA of the cluster in mammalian erythropoiesis, we developed a strategy for stable in vivo individual and combinatorial miRNA inhibition. We developed decoy target sequences for each miRNA expressed by lentiviral vectors marked with distinct fluorescent proteins and used them to probe the functions of miR-144 and miR-451 in the murine hematopoietic system in a competitive repopulation setting. Murine hematopoietic chimeras expressing lentiviral-encoded inhibitory sequences specific for miR-144 or miR-451 exhibited markedly reduced Ter119+ erythroblast counts, with the combined knockdown showing additive effect. These chimeras showed abnormal patterns of erythroid differentiation primarily affecting the proerythroblast to basophilic erythroblast transition, coinciding with the stage where expression of the miRNA cluster is dramatically induced and posttranscriptional gene regulation becomes prominent. These results reveal a role for the miR-144/451 locus in mammalian erythropoiesis and provide the first evidence of functional cooperativity between clustered miRNAs in the hematopoietic system. The strategy described herein will prove useful in functional miRNA studies in mammalian hematopoietic stem cells. STEM CELLS 2010;28:287–296

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