Measurement of cell-penetrating peptide-mediated transduction of adult hematopoietic stem cells.
The ability of cell-penetrating peptides (CPPs) to cross cell membranes and transport cargo into cells makes them an attractive tool for the molecular engineering of stem cells. Even though the exact mechanism of transduction remains unclear, their potential has been demonstrated for diverse applications, including hematopoietic stem cell expansion and the generation of islets cells from embryonic stem cells. Several parameters can affect the intracellular delivery of CPP-based constructs. Those include the type of cells targeted, the type of CPP used, and the properties of the cargo. For this reason, it is important to have a means to quantitatively assess the transduction efficiency of specific constructs in the cell type of interest in order to select the best vector for a specific application. In this chapter, we describe a method to measure the uptake of HIV transactivator of transcription (TAT) and the homeobox protein Antennapedia (Antp) constructs in primary hematopoietic progenitor cells and hematopoietic cell lines. This method is useful to compare, select, and optimize different strategies to deliver CPP-based constructs into a given cell type.
Manceur
Aziza P
AP
Terrence Donnelly Centre for Cellular and Biomolecular Research, Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
Audet
Julie
J
D000328:Adult; D053687:Adult Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002138:Calibration; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D051379:Mice; D018613:Microscopy, Confocal; D010455:Peptides / Q000378:metabolism* / Q000633:toxicity; D018675:Toxicity Tests; D014161:Transduction, Genetic / Q000379:methods*
D010455:Peptides; D016650:Fluorescein-5-isothiocyanate