title: Zinc finger nuclease technology: A stable tool for high efficiency transformation in bloodstream form T. brucei.
abstract: In Trypanosoma brucei, the generation of knockout mutants is relatively easy compared to other organisms as transfection methods are well established. These methods have their limitations, however, when it comes to the generation of genome-wide libraries that require a minimum of several hundred thousand transformants. Double-strand breaks with the meganuclease ISce-I dramatically increase transformation efficiency, but are not widely in use as cell lines need to be generated de novo before each transfection. Here we show that zinc finger nucleases are a robust and stable tool that can enhance transformation in bloodstream forms by more than an order of magnitude.
authors.affiliation: Institute for Cell Biology, Baltzerstrasse 4, 3012 Bern, Switzerland. Electronic address: gabriela.burkard@izb.unibe.ch.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Schumann
authors.affiliation: Institute for Cell Biology, Baltzerstrasse 4, 3012 Bern, Switzerland; Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, Brazil. Electronic address: monicamk@gmail.com.
authors.forename: Monica M
authors.initials: MM
authors.lastname: Kangussu-Marcolino
authors.affiliation: Institute for Cell Biology, Baltzerstrasse 4, 3012 Bern, Switzerland. Electronic address: nicholas.doiron@izb.unibe.ch.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Doiron
authors.affiliation: Institute for Cell Biology, Baltzerstrasse 4, 3012 Bern, Switzerland. Electronic address: sandro.kaeser@dcb.unibe.ch.
authors.forename: Sandro
authors.initials: S
authors.lastname: Käser
authors.affiliation: Institute for Cell Biology, Baltzerstrasse 4, 3012 Bern, Switzerland; Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. Electronic address: gabrielaburle@hotmail.com.
authors.forename: Gabriela
authors.initials: G
authors.lastname: de Assis Burle-Caldas
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, Brazil. Electronic address: wanderson_darocha@yahoo.com.
authors.forename: Wanderson D
authors.initials: WD
authors.lastname: DaRocha
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. Electronic address: santuzat@ufmg.br.
authors.forename: Santuza M
authors.initials: SM
authors.lastname: Teixeira
authors.affiliation: Institute for Cell Biology, Baltzerstrasse 4, 3012 Bern, Switzerland. Electronic address: isabel.roditi@izb.unibe.ch.
authors.forename: Isabel
authors.initials: I
authors.lastname: Roditi
mesh_terms: D003851:Deoxyribonucleases / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D014170:Transformation, Genetic; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D016335:Zinc Fingers
chemical_list: D003851:Deoxyribonucleases
keywords: HA; Trypanosoma brucei; UTR; ZFN; double-strand break; haemagglutinin; homologous recombination; transfection efficiency; untranslated region; zinc finger nuclease; zinc finger nuclease(s)