A new strategy for gene targeting and functional proteomics using the DT40 cell line

A new strategy for gene targeting and functional proteomics using the DT40 cell line

DT40 cells derived from chicken B lymphocytes exhibit exceptionally high homologous recombination rates. Therefore, they can be used as a convenient tool and model for gene targeting experiments. However, lack of efficient cloning strategies, protein purification protocols and a well annotated prote...

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Bibliographic Details
Published in:Nucleic acids research Vol. 41; no. 17; p. e167
Main Authors: Orlowska, Kinga P, Klosowska, Kamila, Szczesny, Roman J, Cysewski, Dominik, Krawczyk, Pawel S, Dziembowski, Andrzej
Format: Journal Article
Language:English
Published: England Oxford University Press 01-09-2013
Subjects:
Animals
Cell Line
Chickens
Cloning, Molecular - methods
Flow Cytometry
Gene Targeting - methods
Genetic Vectors
Methods Online
Microscopy, Fluorescence
Polyribosomes - metabolism
Protein Interaction Mapping
Proteins - analysis
Proteins - genetics
Proteomics - methods
Recombinant Fusion Proteins - analysis
RNA Stability
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Summary:DT40 cells derived from chicken B lymphocytes exhibit exceptionally high homologous recombination rates. Therefore, they can be used as a convenient tool and model for gene targeting experiments. However, lack of efficient cloning strategies, protein purification protocols and a well annotated protein database limits the utility of these cells for proteomic studies. Here we describe a fast and inexpensive experimental pipeline for protein localization, quantification and mass spectrometry-based interaction studies using DT40 cells. Our newly designed set of pQuant vectors and a sequence- and ligation-independent cloning (SLIC) strategy allow for simple and efficient generation of gene targeting constructs, facilitating homologous-recombination-based protein tagging on a multi-gene scale. We also report proof of principle results using the key proteins involved in RNA decay, namely EXOSC8, EXOSC9, CNOT7 and UPF1.
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ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkt650