328 VERSATILE TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE (TALEN)-MEDIATED ENGINEERING OF OSSABAW MINIATURE SWINE

Abstract

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. TALEN-induced double-strand breaks can be used for gene inactivation via repair by nonhomologous end joining (NHEJ) or to stimulate homologous recombination with a user-specified template to create custom-designed disease alleles. Transfection of multiple TALEN pairs targeting the same chromosome can also produce chromosomal rearrangements such as large inversions and deletions. As genetic modification of primary pig fibroblast followed by cloning is the primary method used to generate transgenic swine, we tested our capacity to mimic multiple common mutation types in primary fibroblasts using TALEN. Either plasmids or mRNA encoding TALEN was transfected into 500 000 to 750 000 fibroblasts by nucleofection followed by 3 days of culture at 30°C before analysis of activity. With this approach, we found that over 65% of custom-designed TALEN displayed activity in pig fibroblasts with typical percentage of indel positive chromosomes ranging from 20 to 45%. Individual clones were derived by either co-selection with an unlinked selection transposon or by dilution cloning. Colonies with mono- and biallelic modification to the target loci were readily identified (up to 84 and 24%, respectively) and could be accomplished without the aid of selection. Co-transfection of 2 TALEN pairs directed to the same chromosome resulted in deletion or inversion of the intervening sequence in 10 and 4% of isolated colonies, respectively. In addition, co-transfection of TALEN with a homologous repair template enabled precise insertion of a novel restriction site in nearly 40% of treated cells, 3% of which were homozygous (n = 184 colonies). To prove that modified cells were suitable for generation of animals, we pooled several colonies of Ossabaw fibroblasts harboring TALEN-induced frame-shift mutations in the swine low-density lipoprotein receptor (LDLR) and used them as nuclear donors for cloning. Pregnancy was established in 7/9 transfers (100–150 embryos per transfer), and 6 pregnancies were carried to term, resulting in the live birth of 18 piglets. Three monoallelic frame-shift genotypes were observed among 9 piglets; as expected, each genotype was represented in the parental cell pool. The remaining 9 animals displayed an identical biallelic mutant genotype and are being investigated as a knockout model of familial hypercholesterolemia (FH). Finally, fibroblasts homozygous for a specified inactivating mutation were created by allele introgression and were cloned, demonstrating that gene editing can be used to create precise swine knock-ins in a single generation.