Diagenode

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


Orlowska KP, Klosowska K, Szczesny RJ, Cysewski D, Krawczyk PS, Dziembowski A

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.

Tags
Bioruptor
Cell Lysis
Western Blot

Share this article

Published
September, 2013

Source

Events

  • AAIC
    Los Angeles, CA
    Jul 14-Jul 18, 2019
  • The RNA Society of Japan
    Tokyo
    Jul 17-Jul 19, 2019
 See all events

Applications

 See all applications



The European Regional Development Fund and Wallonia are investing in your future.

Extension of industrial buildings and new laboratories.



  ABOUT SSL CERTIFICATES

         Site map   |   Contact us   |   Conditions of sales   |   Conditions of purchase   |   Privacy policy   |   Diagenode Diagnostics