Abstract
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...Continue Reading
References
Apr 1, 1993·Immunology and Cell Biology·K J StaceyD A Hume
Jan 10, 1997·Experimental Cell Research·M WatanabeN Nakatsuji
Jan 1, 1997·Blood Cells, Molecules & Diseases·V FlochC Férec
Jul 15, 1999·Biochemical and Biophysical Research Communications·H S LiuN J Ke
Aug 6, 1999·Gene Therapy·L H LiS W Hui
Feb 26, 2000·Science·I L Weissman
Mar 29, 2000·Biochemical and Biophysical Research Communications·T ShimokawaC Ra
Sep 12, 2000·Gene Therapy·V F Van TendelooZ N Berneman
Mar 22, 2001·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·L H LiS W Hui
Nov 20, 2001·Stem Cells·M H WuM E Dolan
May 29, 2002·Tissue Engineering·Astrid HammAnja K Bosserhoff
Oct 22, 2002·Nature Immunology·Julie Harriague, Georges Bismuth
Jan 4, 2003·Blood·Christopher BaumChristof von Kalle
Feb 11, 2003·The Journal of Laboratory and Clinical Medicine·Florian WeissingerMartin Wilhelm
Mar 1, 2003·Journal of Immunological Methods·Hans-Ingo TrompeterMarkus Uhrberg
Mar 1, 2003·Nature Genetics·Kathryn V Anderson, Philip W Ingham
Apr 30, 2003·Human Gene Therapy·Cordula LeursHelmut Hanenberg
Dec 1, 1963·British Journal of Cancer·E H PORTER, R J BERRY
May 4, 2004·Methods : a Companion to Methods in Enzymology·Oliver GreschKlaus Lun
Jul 3, 2004·Blood·Bernd GiebelPeter Wernet
Jul 28, 2004·Stem Cells·Uma LakshmipathyCatherine M Verfaillie
Sep 21, 2004·Oncogene·Guy SauvageauR Keith Humphries
Nov 2, 2004·Nature Reviews. Immunology·Brian P Sorrentino
Citations
Jun 25, 2009·Molecular Imaging and Biology : MIB : the Official Publication of the Academy of Molecular Imaging·Feng CaoJoseph C Wu
Jan 1, 2010·Biochemistry·Jianhui ZhuClifford J Steer
Aug 5, 2010·The Journal of Biological Chemistry·Ana-Violeta FonsecaDenis Corbeil
Aug 20, 2009·Human Gene Therapy·Teiko SumiyoshiDonald B Kohn
Aug 23, 2008·Nucleic Acids Research·Christof MauckschCarsten Rudolph
Feb 8, 2011·Respiratory Research·Lunyin Yu, Charles A Hales
Jul 12, 2008·Stem Cells·Sören MoritzAlexander von Holst
Sep 1, 2007·Platelets·Caroline PendariesJennifer C Spalton
Oct 25, 2011·Experimental Hematology·Inho KimAndre Larochelle
Nov 23, 2010·Journal of Biotechnology·Catarina MadeiraJoaquim M S Cabral
Jan 22, 2014·Stem Cells·Viraj KamatScott L Diamond
Nov 26, 2015·Scientific Reports·Yvonne DienerAndreas Bosio
Mar 31, 2015·British Journal of Haematology·Stefan RadtkeBernd Giebel
Feb 27, 2013·The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology·Woosuk KimHyun-Jung Kim
Oct 27, 2016·Experimental Hematology·Doreen ReichertDenis Corbeil
Oct 27, 2016·Experimental Hematology·Yvonne DienerUte Bissels
Oct 4, 2016·Therapeutic Advances in Hematology·Michael A Goodman, Punam Malik
Feb 27, 2016·F1000Research·Astrid GlaserJim Vadolas
Aug 21, 2020·Proceedings of the National Academy of Sciences of the United States of America·Ludwig SchmidererMartin Hjort
Mar 14, 2021·Gene Therapy·Colin L SweeneySuk See De Ravin