Document Detail


Pooling and PCR as a method to combat low frequency gene targeting in mouse embryonic stem cells.
MedLine Citation:
PMID:  19002898     Owner:  NLM     Status:  PubMed-not-MEDLINE    
Abstract/OtherAbstract:
The introduction of germ line modifications by gene targeting in mouse embryonic stem (ES) cells has proven a fundamental technology to relate genes to mammalian biology. Critical aspects required for successful gene targeting have traditionally been experimental enhancements that increase the frequency or detection of homologous recombination within ES cells; however, the utilization of such methods may still result in the failed isolation of a positively targeted ES cell clone. In this study, we discuss the current enhancement methods and describe an ES cell pooling strategy that maximizes the ability to detect properly targeted ES cells regardless of an inherent low targeting efficiency. The sensitivity required to detect correctly targeted events out of a pool of ES cell clones is provided by polymerase chain reaction (PCR), and only those pools containing positives need to be expanded and screened to find individually targeted clones. This method made it possible to identify targeted clones from a screen of approximately 2,300 ES cell colonies by performing only 123 PCR reactions. This technically streamlined approach bypasses the need to troubleshoot and re-engineer an existing targeting construct that is functionally suitable despite its low targeting frequency.
Authors:
A C Brown; C P Lerner; J H Graber; D J Shaffer; D C Roopenian
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Publication Detail:
Type:  Journal Article     Date:  2006-11-02
Journal Detail:
Title:  Cytotechnology     Volume:  51     ISSN:  0920-9069     ISO Abbreviation:  Cytotechnology     Publication Date:  2006 Jun 
Date Detail:
Created Date:  2008-11-12     Completed Date:  2011-07-14     Revised Date:  2013-02-19    
Medline Journal Info:
Nlm Unique ID:  8807027     Medline TA:  Cytotechnology     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  81-8     Citation Subset:  -    
Affiliation:
The Jackson Laboratory, 600 Main Street, Bar Harbor, ME, 04609, USA, aaron.brown@jax.org.
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