Production of myostatin gene knockout Wuzhishan miniature pig fibroblasts with zinc-finger nucleases

Abstract

Disruption of myostatin (MSTN) gene in pigs may improve porcine lean meat percentage (LMP), and create an animal model for certain human diseases. Using zinc-finger nucleases (ZFNs) technology, MSTN gene was deleted in Wuzhishan miniature pig fibroblasts by transfection of either ZFNs plasmids or ZFNs mRNA in high efficiency. Strikingly, ZFNs encoding mRNA could produce MSTN+/-and MSTN-/- cell colonies with single or double allele deletion by single transfection. Sequencing results demonstrated that 92.18% of the mutations were short fragment deletions or insertions (≤10 bp). Prediction of amino acids sequences indicated that more than half of the mutations cause premature transla-tional-termination codon. MSTN+/+, MSTN+/-, and MSTN-/- cell colonies were used as nuclear donor for somatic cell nuclear transfer (SCNT), and developmental potential of SCNT embryos were measured by the blastocyst rate. The results revealed no significant difference in development competence among the three kinds of reconstructed embryos (14.29% vs. 19.64% vs. 16.13%), which provides the possibility of making myostatin knock out pigs in the future.

Targeted Mutagenesis of Beta-Lactoglobulin Gene in Caprine Fetal Fibroblasts by Context-Dependent Assembly Zinc-Finger Nucleases

“…All mutations were centered on the ZFN cleavage site, and were consistent with intron 3. The types of mutation recovered were consistent with those already reported, such as deletions, substitutions and insertions [8] [9] [13] [17]- [20]. To test whether ZFN plasmid randomly integrated into the goat genome, PCR was performed to examine ZFN plasmid integration by amplifying CMV-FokI catalytic domain from all mutant clones.…”