| Nucleofection, an efficient nonviral method to transfer genes into human hematopoietic stem and progenitor cells. | |
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MedLine Citation:
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PMID: 16646674 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The targeted manipulation of the genetic program of single cells as well as of complete organisms has strongly enhanced our understanding of cellular and developmental processes and should also help to increase our knowledge of primary human stem cells, e.g., hematopoietic stem cells (HSCs), within the next few years. An essential requirement for such genetic approaches is the existence of a reliable and efficient method to introduce genetic elements into living cells. Retro- and lentiviral techniques are efficient in transducing primary human HSCs, but remain labor and time consuming and require special safety conditions, which do not exist in many laboratories. In our study, we have optimized the nucleofection technology, a modified electroporation strategy, to introduce plasmid DNA into freshly isolated human HSC-enriched CD34(+) cells. Using enhanced green fluorescent protein (eGFP)-encoding plasmids, we obtained transfection efficiencies of approximately 80% and a mean survival rate of 50%. Performing functional assays using GFU-GEMM and long-term culture initiating cells (LTC-IC), we demonstrate that apart from a reduction in the survival rate the nucleofection method itself does not recognizably change the short- or long-term cell fate of primitive hematopoietic cells. Therefore, we conclude, the nucleofection method is a reliable and efficient method to manipulate primitive hematopoietic cells genetically. |
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Authors:
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Gregor von Levetzow; Jan Spanholtz; Julia Beckmann; Johannes Fischer; Gesine Kögler; Peter Wernet; Michael Punzel; Bernd Giebel |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Stem cells and development Volume: 15 ISSN: 1547-3287 ISO Abbreviation: Stem Cells Dev. Publication Date: 2006 Apr |
Date Detail:
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Created Date: 2006-05-01 Completed Date: 2006-07-10 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 101197107 Medline TA: Stem Cells Dev Country: United States |
Other Details:
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Languages: eng Pagination: 278-85 Citation Subset: IM |
Affiliation:
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Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University Düsseldorf, D-40225 Düsseldorf, Germany. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Chloromethyl Ketones
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pharmacology Animals Antigens, CD / analysis Antigens, CD34 / analysis Cell Culture Techniques / methods Cell Line Cell Proliferation / drug effects Cell Separation Cell Survival / drug effects Colony-Forming Units Assay Electroporation / methods Erythrocytes / cytology, metabolism Fetal Blood / cytology Flow Cytometry Glycoproteins / analysis Green Fluorescent Proteins / genetics, metabolism Hematopoietic Stem Cells / chemistry, cytology, metabolism* Humans Leukocytes / cytology, metabolism Microscopy, Fluorescence Peptides / analysis Stromal Cells / cytology Transfection / methods* |
| Chemical | |
Reg. No./Substance:
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0/AC133 antigen; 0/Amino Acid Chloromethyl Ketones; 0/Antigens, CD; 0/Antigens, CD34; 0/Glycoproteins; 0/Peptides; 0/benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; 147336-22-9/Green Fluorescent Proteins |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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