mRNA transfection of CXCR4-GFP fusion--simply generated by PCR-results in efficient migration of primary human mesenchymal stem cells

Tissue Eng Part C Methods. 2008 Sep;14(3):179-84. doi: 10.1089/ten.tec.2007.0359.


We present a general, entirely PCR-based strategy to construct mRNAs coding for green fluorescent protein (GFP) fusion proteins from a cDNA pool. We exemplify our approach for the chemokine receptor CXCR4. mRNA transfection of the PCR-generated fusion of CXCR4-GFP into K562 cells or primary mesenchymal stem cells (MSCs) resulted in excellent viability (> 90%) with more than 90% of target cells expressing easily detectable CXCR4-GFP for > 72 h. The fusion protein was localized in the plasma membrane and was rapidly internalized upon incubation with the CXCR4 ligand stromal cell-derived factor-1 (SDF-1). Transwell migration experiments showed significantly increased migration of CXCR4-GFP mRNA-transfected MSCs toward a gradient of SDF-1, demonstrating that mRNA-mediated chemokine receptor overexpression allows for transient initiation of chemotaxis. The presented strategy to construct a PCR-based fluorescent fusion protein can be generally applied to other genes of interest to study their function by simple overexpression and easy detection in primary cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bone Marrow Cells / metabolism
  • Cell Movement
  • Chemokine CXCL12 / metabolism
  • Chemotaxis
  • Green Fluorescent Proteins / metabolism
  • Humans
  • K562 Cells
  • Ligands
  • Mesenchymal Stem Cells / cytology*
  • Models, Genetic
  • Polymerase Chain Reaction
  • RNA, Messenger / metabolism*
  • Receptors, CXCR4 / metabolism*
  • Tissue Engineering / methods*
  • Transfection


  • CXCL12 protein, human
  • CXCR4 protein, human
  • Chemokine CXCL12
  • Ligands
  • RNA, Messenger
  • Receptors, CXCR4
  • Green Fluorescent Proteins