Use of a micromanipulator for high-efficiency cloning of cells co-expressing fluorescent proteins

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DOIResolve DOI: http://doi.org/10.1023/A:1009871029495
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TypeArticle
Journal titleMethods in Cell Science
Volume22
Issue2
Pages137145; # of pages: 9
AbstractThe inclusion of the gene encoding the Green Fluorescent Protein (GFP) or its derivatives into dicistronic transfer vectors is a useful method to visually identify cells that have incorporated a specific gene of interest. By combining this approach with the use of a micromanipulator, we have developed a protocol for the one-step isolation of cells expressing a specific transgene from a pool of transfected cells. Target fluorescent cells could be identified and isolated even when they occured at frequencies as low as 1/100,000. The use of Leibowitz L-15 serum-free medium and serum-coated non-charged petri dishes, along with minimal light exposure yielded maximal cell viability and high cloning efficiency (≈ 40%, on average) for a large number of cell lines, both adherent and suspension. Several variations of the basic method are presented, as well as guidelines for the choice of hardware components to implement our cloning workstation.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada; NRC Biotechnology Research Institute
Peer reviewedNo
NPARC number12328573
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Record identifier473dcc43-7712-4af7-9935-dea233602ae9
Record created2009-09-10
Record modified2016-05-09
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