title: Efficient TALEN-mediated gene knockout in livestock.
abstract: Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.
authors.affiliation: Center for Genome Engineering and Department of Animal Science, University of Minnesota, St Paul, MN 55108, USA.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Carlson
authors.forename: Wenfang
authors.initials: W
authors.lastname: Tan
authors.forename: Simon G
authors.initials: SG
authors.lastname: Lillico
authors.forename: Dana
authors.initials: D
authors.lastname: Stverakova
authors.forename: Chris
authors.initials: C
authors.lastname: Proudfoot
authors.forename: Michelle
authors.initials: M
authors.lastname: Christian
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Voytas
authors.forename: Charles R
authors.initials: CR
authors.lastname: Long
authors.forename: C Bruce A
authors.initials: CB
authors.lastname: Whitelaw
authors.forename: Scott C
authors.initials: SC
authors.lastname: Fahrenkrug
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002872:Chromosome Deletion; D007446:Chromosome Inversion; D019976:Cloning, Organism; D004247:DNA; D004251:DNA Transposable Elements; D055786:Gene Knockout Techniques; D058751:Livestock / Q000235:genetics*; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D013552:Swine; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements; D014157:Transcription Factors; D004247:DNA