Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases.

Zinc-finger nucleases (ZFNs) are powerful tools for producing gene knockouts (KOs) with high efficiency. Whereas ZFN-mediated gene disruption has been demonstrated in laboratory animals such as mice, rats, and fruit flies, ZFNs have not been used to disrupt an endogenous gene in any large domestic species. Here we used ZFNs to induce a biallelic knockout of the porcine α1,3-galactosyltransferase (GGTA1) gene. Primary porcine fibroblasts were treated with ZFNs designed against the region coding for the catalytic core of GGTA1, resulting in biallelic knockout of ∼1% of ZFN-treated cells. A galactose (Gal) epitope counter-selected population of these cells was used in somatic cell nuclear transfer (SCNT). Of the resulting six fetuses, all completely lacked Gal epitopes and were phenotypically indistinguishable from the starting donor cell population, illustrating that ZFN-mediated genetic modification did not interfere with the cloning process. Neither off-target cleavage events nor integration of the ZFN-coding plasmid was detected. The GGTA1-KO phenotype was confirmed by a complement lysis assay that demonstrated protection of GGTA1-KO fibroblasts relative to wild-type cells. Cells from GGTA1-KO fetuses and pooled, transfected cells were used to produce live offspring via SCNT. This study reports the production of cloned pigs carrying a biallelic ZFN-induced knockout of an endogenous gene. These findings open a unique avenue toward the creation of gene KO pigs, which could benefit both agriculture and biomedicine.

Identifying Fishes through DNA Barcodes and Microarrays.

BACKGROUND: International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. METHODOLOGY/PRINCIPAL FINDINGS: This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of "DNA barcoding" and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the "position of label" effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. CONCLUSIONS/SIGNIFICANCE: Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.