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Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells
B R Davis1, J Yannariello-Brown, N L Prokopishyn
1Sealy Center for Oncology and Hematology, Department of Microbiology and Immunology, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555-0645, USA.
Abstract:
A novel glass needle-mediated microinjection method for delivery of macromolecules, including proteins and larger transgene DNAs, into the nuclei of blood stem/progenitor cells was developed. Temporary immobilization of cells to extracellular matrix-coated dishes has enabled rapid and consistent injection of macromolecules into nuclei of CD34(+), CD34(+)/CD38(-), and CD34(+)/CD38(-)/Thy-1(lo) human cord blood cells. Immobilization and detachment protocols were identified, which had no adverse effect on cell survival, progenitor cell function (colony forming ability), or stem cell function (NOD/SCID reconstituting ability). Delivery of fluorescent dextrans to stem/progenitor cells was achieved with 52% +/- 8.4% of CD34(+) cells and 42% +/- 14% of CD34(+)/CD38(-)cells still fluorescent 48 hours after injection. Single-cell transfer and culture of injected cells has demonstrated long-term survival and proliferation of CD34(+) and CD34(+)/CD38(-) cells, and retention of the ability of CD34(+)/CD38(-) cells to generate progenitor cells. Delivery of DNA constructs (currently