337 NONMEIOTIC INTROGRESSION OF QUANTITATIVE TRAIT NUCLEOTIDES AND CORRECTION OF CONGENITAL MUTATIONS IN LIVESTOCK WITH TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES
S. C. Fahrenkrug A C , W. Tan B C , S. G. Lillico D , D. Stverakova D , C. Proudfoot D , G. Williamson E , C. R. Long E , C. B. A. Whitelaw D and D. F. Carlson AA Recombinetics, St. Paul, MN, USA;
B Department of Animal Science, University of Minnesota, St. Paul, MN, USA;
C Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA;
D The Roslin Institute and R(D)SVS, University of Edinburgh, Easter Bush Campus, Midlothian, United Kingdom;
E Veterinary Physiology & Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX, USA
Reproduction, Fertility and Development 25(1) 316-316 https://doi.org/10.1071/RDv25n1Ab337
Published: 4 December 2012
Abstract
Genetic enhancement of livestock productivity and welfare are major goals of breeding and genetics programs. However, the introgression of desirable alleles across breeds is slow and inaccurate. The development of gene editing technologies would provide the opportunity to accelerate the genetic improvement of a diversity of livestock breeds. Transcription activator-like effector nucleases (TALEN) are programmable nucleases that join the modular DNA binding domain of transcription activator-like (TAL) effectors with FokI endonuclease. We found that TALEN could be easily manufactured and that 64% displayed activity in swine and cattle primary fibroblasts, with cleavage of 1.5 to 45% of chromosomes in cell populations, as measured by Surveyor assay. Clonal isolation and sequencing revealed that up to 84% of cells contained at least one modified allele, with up to 24% of cells containing biallelic or homozygous chromosomal modification. Co-transfection of a customized TALEN pair with a template containing a specific allele was effective at the nonmeiotic introgression of quantitative trait into naïve cattle breeds. We will also describe the repair of 2 recently described embryonic lethal mutations that are segregating in important dairy cattle breeds (JH1 and HH1). Injection of TALEN mRNA into the cytoplasm of pig and cattle zygotes was capable of inducing gene knockout (KO) in 27 to 75% of embryos analysed (n = 4–59), nearly half of which (8/19) harbored biallelic modification. We will present data describing efforts towards gene conversion by direct injection of livestock embryos. Finally, we will present alternative strategies for the incorporation of gene editing in livestock production systems by cloning or embryo treatment.
