title: Conditionals by inversion provide a universal method for the generation of conditional alleles.
abstract: Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene's transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon-intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN's artificial intron opens up engineering modalities for the generation of multifunctional alleles.
authors.affiliation: Regeneron Pharmaceuticals, Inc, Tarrytown, NY 10591, USA. aris@regeneron.com
authors.forename: Aris N
authors.initials: AN
authors.lastname: Economides
authors.forename: David
authors.initials: D
authors.lastname: Frendewey
authors.forename: Peter
authors.initials: P
authors.lastname: Yang
authors.forename: Melissa G
authors.initials: MG
authors.lastname: Dominguez
authors.forename: Anthony T
authors.initials: AT
authors.lastname: Dore
authors.forename: Ivan B
authors.initials: IB
authors.lastname: Lobov
authors.forename: Trikaldarshi
authors.initials: T
authors.lastname: Persaud
authors.forename: Jose
authors.initials: J
authors.lastname: Rojas
authors.forename: Joyce
authors.initials: J
authors.lastname: McClain
authors.forename: Peter
authors.initials: P
authors.lastname: Lengyel
authors.forename: Gustavo
authors.initials: G
authors.lastname: Droguett
authors.forename: Rostislav
authors.initials: R
authors.lastname: Chernomorsky
authors.forename: Sean
authors.initials: S
authors.lastname: Stevens
authors.forename: Wojtek
authors.initials: W
authors.lastname: Auerbach
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Dechiara
authors.forename: William
authors.initials: W
authors.lastname: Pouyemirou
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Cruz
authors.forename: Kieran
authors.initials: K
authors.lastname: Feeley
authors.forename: Ian A
authors.initials: IA
authors.lastname: Mellis
authors.forename: Jason
authors.initials: J
authors.lastname: Yasenchack
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Hatsell
authors.forename: Liqin
authors.initials: L
authors.lastname: Xie
authors.forename: Esther
authors.initials: E
authors.lastname: Latres
authors.forename: Lily
authors.initials: L
authors.lastname: Huang
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Evangelos
authors.initials: E
authors.lastname: Pefanis
authors.forename: Dimitris
authors.initials: D
authors.lastname: Skokos
authors.forename: Ron A
authors.initials: RA
authors.lastname: Deckelbaum
authors.forename: Susan D
authors.initials: SD
authors.lastname: Croll
authors.forename: Samuel
authors.initials: S
authors.lastname: Davis
authors.forename: David M
authors.initials: DM
authors.lastname: Valenzuela
authors.forename: Nicholas W
authors.initials: NW
authors.lastname: Gale
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Murphy
authors.forename: George D
authors.initials: GD
authors.lastname: Yancopoulos
mesh_terms: D000483:Alleles; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D020868:Gene Silencing; D005818:Genetic Engineering / Q000379:methods*; D016254:Mutagenesis, Insertional / Q000379:methods*; D057345:Sequence Inversion / Q000235:genetics*
chemical_list: D004254:DNA Nucleotidyltransferases; C077570:Site-specific recombinase
keywords: conditional-null; genome engineering
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