Glass needle–mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells
| Autor: | Jue Wang, Nicole L. Prokopishyn, Judith Yannariello-Brown, Mark R. Smith, David B. Brown, Brian R. Davis, N. D. Victor Carsrud, Zhongjun Luo |
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| Rok vydání: | 2000 |
| Předmět: | |
| Zdroj: | Blood. 95:437-444 |
| ISSN: | 1528-0020 0006-4971 |
| DOI: | 10.1182/blood.v95.2.437 |
| Popis: | 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 CD341, CD341/CD382, and CD341/CD382/Thy-1lo 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% 6 8.4% of CD341 cells and 42% 6 14% of CD341/CD382cells still fluorescent 48 hours after injection. Single-cell transfer and culture of injected cells has demonstrated long-term survival and proliferation of CD341 and CD341/CD382 cells, and retention of the ability of CD341/ CD382 cells to generate progenitor cells. Delivery of DNA constructs (currently I 19.6 kb) and fluorescently labeled proteins into CD34 1 and CD34 1 /CD38 2 cells was achieved with transient expression of green fluorescent protein observed in up to 75% of injected cells. These data indicate that glass needle‐mediated delivery of macromolecules into primitive hematopoietic cells is a valuable method for studies of stem cell biology and a promising method for human blood stem cell gene therapy. (Blood 2000;95:437-444) |
| Databáze: | OpenAIRE |
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