Document Detail


New technique for gene transfection using laser irradiation.
MedLine Citation:
PMID:  11288759     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
BACKGROUND: We have developed a gene transfection system using laser beams. The principle of this procedure is that a small hole is made in a cell membrane by pulse laser irradiation, and a gene contained in a medium is transferred into the cytoplasm through the hole. This hole disappears immediately with the application of laser irradiation of the appropriate power. METHODS: A pulse-wave Nd:YAG laser with a wavelength of 355 nm was used to make a hole in a cell membrane. To trap a cell, a continuous-wave Nd:YAG laser with a wavelength of 1015 nm was used. Plasmids that encode the enhanced green fluorescent protein (EGFP) gene were contained in a medium and transferred to HuH-7 and NIH/3T3 cells with pulse laser irradiation. We evaluated transfection efficiency on the basis of the number of cells that expressed EGFP. Stimulatory protein 2 cells in suspension were fixed using a trapping laser and the neomycin-resistance gene was transfected by pulse laser irradiation. We examined cell proliferation in the selection medium. RESULTS: Cells that expressed EGFP were recognized in the group that was irradiated by pulse laser. No cells expressed EGFP without irradiation. Transfection efficiency was approximately 10% at a plasmid concentration of 10.0 microg/mL. At concentrations greater than 20 microg/mL, the transfection rate reached a plateau. We also successfully transfected neomycin-resistance genes to cells floating in suspension after fixation that was achieved with trapping laser irradiation. CONCLUSIONS: This method enables us to transfect targeted cells, ie, cells in suspension as well as attached cells, with a simple technique that does not involve harmful vectors. The present method is very useful for gene transfection in cellular biotechnology.
Authors:
Y Shirahata; N Ohkohchi; H Itagak; S Satomi
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Journal of investigative medicine : the official publication of the American Federation for Clinical Research     Volume:  49     ISSN:  1081-5589     ISO Abbreviation:  J. Investig. Med.     Publication Date:  2001 Mar 
Date Detail:
Created Date:  2001-04-05     Completed Date:  2001-05-31     Revised Date:  2006-05-01    
Medline Journal Info:
Nlm Unique ID:  9501229     Medline TA:  J Investig Med     Country:  United States    
Other Details:
Languages:  eng     Pagination:  184-90     Citation Subset:  IM    
Affiliation:
Division of Advanced Surgical Science and Technology, Graduate School of Medicine, Tohoku University, Sendai, Japan. sirahata@mail.cc.tohoku.ac.jp
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MeSH Terms
Descriptor/Qualifier:
3T3 Cells
Animals
Green Fluorescent Proteins
Luminescent Proteins / genetics
Mice
Mice, Inbred BALB C
Plasmids
Transfection / methods*
Chemical
Reg. No./Substance:
0/Luminescent Proteins; 147336-22-9/Green Fluorescent Proteins

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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