Document Detail


Nucleofection, an efficient nonviral method to transfer genes into human hematopoietic stem and progenitor cells.
MedLine Citation:
PMID:  16646674     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Gregor von Levetzow; Jan Spanholtz; Julia Beckmann; Johannes Fischer; Gesine Kögler; Peter Wernet; Michael Punzel; Bernd Giebel
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Stem cells and development     Volume:  15     ISSN:  1547-3287     ISO Abbreviation:  Stem Cells Dev.     Publication Date:  2006 Apr 
Date Detail:
Created Date:  2006-05-01     Completed Date:  2006-07-10     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  101197107     Medline TA:  Stem Cells Dev     Country:  United States    
Other Details:
Languages:  eng     Pagination:  278-85     Citation Subset:  IM    
Affiliation:
Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine-University Düsseldorf, D-40225 Düsseldorf, Germany.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Chloromethyl Ketones / 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:
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

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