240 PRECISE GENOME EDITING OF PDX1 BY DIRECT INJECTION OF TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES (TALENS) INTO PARTHENOGENETIC PIG EMBRYOS

Abstract

Pancreatic and duodenal homeobox 1 (PDX1) is one of the transcription factors involved in pancreatic organogenesis and plays a critical role as an early lineage marker of pancreatic specification and β-cell differentiation. In mature pancreas, PDX1 regulates a large number of genes involved in maintaining β-cell identity and function. In mice and humans, its homozygous disruption results in pancreas agenesis, while heterozygous mutations have been associated with early-onset (MODY) and late-onset forms of Type II diabetes mellitus in humans. Knockout of the PDX1 gene in pigs may lead to the generation of an apancreatic phenotype, which in turn could allow the potential generation of an exogenic pancreas. Moreover, this could help to create a large animal model for human diabetes. We used transcription activator-like effector nucleases (TALEN) technology with the aim of studying the efficiency of precise editing by homologous recombination in parthenogenetically activated porcine embryos. Mature oocytes were activated by incubation in ionomycin (10 µM) for 20 min, followed by a 4-h incubation in DMAP (2 mM) + cytochalasin B (7.5 µg mL^–1). Cytoplasmic injection was performed 14 h post-activation. Post injections, embryos were cultured for 6 days in NCSU23+BSA media at 38.5°C in a 5% CO₂ atmosphere. We first evaluated the activity of a pair of TALENs targeting the functional domain of the PDX1 gene. Three concentrations of mRNA were microinjected (10, 20, and 40 ng µL^–1) and blastocysts were analysed for non-homologous end-joining (NHEJ) by sequencing. The efficiency of indel mutations at the PDX1-target loci (either monoallelic or biallelic) was 34.5, 52.6, and 80.0% for the 10, 20, and 40 ng µL^–1 concentrations, respectively. Next, we tested whether ssODNs (single-stranded oligodeoxynucleotide) coinjected with TALENs would permit precise homology direct repair (HDR) in porcine parthenotes. TALEN mRNA (40 ng µL^–1) was coinjected with an ssODN donor template (50 ng µL^–1) designed to incorporate a novel stop codon, HindIII restriction site, and a frame shift mutation. Cleavage and blastocyst rates were recorded at Days 2 and 6 of development, respectively, in the TALEN/ssODN injected group (n = 260 zygotes), buffer-injected embryos (n = 135 zygotes), and the non-injected group (n = 132 zygotes). Day 7 embryos were analysed for NHEJ and HDR by RFLP assay and Sanger sequencing after whole-genome amplification and PCR. Blastocyst rates were 15% (TALEN/ssODN-injected group), 27% (buffer-injected group), and 34% (non-injected group). A total of 30 blastocysts were analysed for HDR after whole-genome amplification. The majority of analysed blastocysts (28/30, 93%) were mutant. Among them, 10 (36%) incorporated the ssODN, from which 3 (30%) showed a KO genotype with a precise biallelic modification. We report here a highly efficient and precise TALEN-mediated gene knockout in swine embryos, which represents an alternative to cloning for phenotype evaluation of knockouts related to organogenesis.