mRNA transfection of CXCR4-GFP fusion--simply generated by PCR-results in efficient migration of primary human mesenchymal stem cells.
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.
Ryser
Martin F
MF
Department of Pediatrics, University Clinic Dresden, Dresden, Germany.
Ugarte
Fernando
F
Thieme
Sebastian
S
Bornhäuser
Martin
M
Roesen-Wolff
Angela
A
Brenner
Sebastian
S
D001854:Bone Marrow Cells / Q000378:metabolism; D002465:Cell Movement; D054377:Chemokine CXCL12 / Q000378:metabolism; D002633:Chemotaxis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D008024:Ligands; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D019718:Receptors, CXCR4 / Q000378:metabolism*; D023822:Tissue Engineering / Q000379:methods*; D014162:Transfection
C515255:CXCL12 protein, human; C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; D008024:Ligands; D012333:RNA, Messenger; D019718:Receptors, CXCR4; D049452:Green Fluorescent Proteins