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.