title: A new strategy for gene targeting and functional proteomics using the DT40 cell line.
abstract: 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.
authors.affiliation: Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Pawinskiego 5a, 02-106, Warsaw, Poland and Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland.
authors.forename: Kinga P
authors.initials: KP
authors.lastname: Orlowska
authors.forename: Kamila
authors.initials: K
authors.lastname: Klosowska
authors.forename: Roman J
authors.initials: RJ
authors.lastname: Szczesny
authors.forename: Dominik
authors.initials: D
authors.lastname: Cysewski
authors.forename: Pawel S
authors.initials: PS
authors.lastname: Krawczyk
authors.forename: Andrzej
authors.initials: A
authors.lastname: Dziembowski
mesh_terms: D000818:Animals; D002460:Cell Line; D002645:Chickens; D003001:Cloning, Molecular / Q000379:methods*; D005434:Flow Cytometry; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D008856:Microscopy, Fluorescence; D011132:Polyribosomes / Q000378:metabolism; D025941:Protein Interaction Mapping; D011506:Proteins / Q000032:analysis / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D020871:RNA Stability; D011993:Recombinant Fusion Proteins / Q000032:analysis
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins
references.citation: Nature. 2003 Oct 16;425(6959):686-91
references.pmid: 14562095
references.citation: EMBO J. 2010 Jul 21;29(14):2342-57
references.pmid: 20531386
references.citation: Nat Biotechnol. 2011 May 15;29(7):644-52
references.pmid: 21572440
references.citation: EMBO J. 1994 Feb 1;13(3):595-605
references.pmid: 8313904
references.citation: J Vis Exp. 2010 Jun 15;(40):
references.pmid: 20567211
references.citation: Nat Biotechnol. 2008 Dec;26(12):1367-72
references.pmid: 19029910
references.citation: Nature. 2002 Jan 10;415(6868):180-3
references.pmid: 11805837
references.citation: Methods Mol Biol. 2007;408:193-210
references.pmid: 18314584
references.citation: Mol Cell. 2011 Aug 19;43(4):624-37
references.pmid: 21855801
references.citation: Nat Methods. 2011 Jan;8(1):67-9
references.pmid: 21151135
references.citation: Mol Cell. 2004 Aug 27;15(4):607-20
references.pmid: 15327776
references.citation: EMBO J. 1998 Feb 16;17(4):1096-106
references.pmid: 9463387
references.citation: Nucleic Acids Res. 1997 Feb 15;25(4):814-21
references.pmid: 9064659
references.citation: Subcell Biochem. 2006;40:419-21
references.pmid: 17623933
references.citation: Mol Biol Cell. 1995 May;6(5):611-25
references.pmid: 7545033
references.citation: Nat Methods. 2009 Mar;6(3):203-5
references.pmid: 19198594
references.citation: Cell. 2012 Aug 17;150(4):855-66
references.pmid: 22901814
references.citation: Nature. 2003 Oct 16;425(6959):737-41
references.pmid: 14562106
references.citation: Nat Methods. 2011 Jan;8(1):74-9
references.pmid: 21131970
references.citation: Nature. 2004 Dec 9;432(7018):695-716
references.pmid: 15592404
references.citation: Science. 2002 Feb 15;295(5558):1301-6
references.pmid: 11847344
references.citation: J Proteome Res. 2006 Dec;5(12):3446-52
references.pmid: 17137347
references.citation: Virology. 1985 Jul 15;144(1):139-51
references.pmid: 2998040
references.citation: Cell. 1991 Oct 4;67(1):179-88
references.pmid: 1913816
references.citation: J Proteome Res. 2011 Apr 1;10(4):1794-805
references.pmid: 21254760
references.citation: Nature. 2006 Mar 30;440(7084):637-43
references.pmid: 16554755
references.citation: Genes Dev. 2006 Feb 1;20(3):355-67
references.pmid: 16452507
references.citation: EMBO J. 1990 Mar;9(3):921-7
references.pmid: 2155784
references.citation: Nature. 2002 Jan 10;415(6868):141-7
references.pmid: 11805826
references.citation: Nature. 2006 Mar 30;440(7084):631-6
references.pmid: 16429126
references.citation: Biochem J. 2003 Aug 1;373(Pt 3):775-83
references.pmid: 12723973
references.citation: Nat Struct Mol Biol. 2005 Dec;12(12):1054-63
references.pmid: 16284618
references.citation: Nat Methods. 2012 Jun;9(6):588-90
references.pmid: 22543349
references.citation: Nat Methods. 2007 Mar;4(3):251-6
references.pmid: 17293868
references.citation: Mol Cell Biol. 2005 Mar;25(5):1958-70
references.pmid: 15713649
references.citation: RNA Biol. 2013 Feb;10(2):228-44
references.pmid: 23303381
references.citation: Cell. 2000 Dec 22;103(7):1121-31
references.pmid: 11163187
references.citation: Nat Methods. 2013 Apr;10(4):315-23
references.pmid: 23435261
references.citation: Mol Cell Biol. 2000 Dec;20(23):8944-57
references.pmid: 11073994
references.citation: Anal Biochem. 1999 Feb 15;267(2):344-50
references.pmid: 10036140
references.citation: Science. 2002 Oct 11;298(5592):419-22
references.pmid: 12228722
references.citation: BMC Biotechnol. 2001;1:7
references.pmid: 11591226
references.citation: Mol Cell Biol. 1998 Nov;18(11):6430-5
references.pmid: 9774659
references.citation: Nucleic Acids Res. 2012 Apr;40(8):e55
references.pmid: 22241772
references.citation: J Biol Chem. 2000 May 26;275(21):16191-201
references.pmid: 10748092
references.citation: Nat Struct Mol Biol. 2005 Sep;12(9):763-71
references.pmid: 16116434