Characterization of <i>MSTN</i> gene knockout in cloned cattle.

<p>(A) Sequencing analysis of <i>MSTN</i> biallelic mutation types in cloned calves. The <i>MSTN</i> biallelic mutation types consisted of a 6-bp deletion in one allele (the last 4 bp of exon 1 and the first 2 bp of intron 1) and a 117-bp deletion (nucleotide positions 8–124 in intron 1) and a 9-bp insertion (the last 2 bp of exon 1 and the first 7 bp of intron 1, AG GCACGGG) in the other allele, which were consistent with colony 6. The red letters represent <i>MSTN</i> exon 1, and the blue letters represent <i>MSTN</i> intron 1. (B) Calves with <i>MSTN</i> biallelic mutations displayed the doubled-muscled phenotype, and exhibited no untoward effects. In red circles, the muscle mass in <i>MSTN</i> mutant (left) was greater than that of the wild-type calf (right). (C) Hematoxylin and eosin -stained cross-sections of the quadriceps muscle. Muscle fibers from calves with <i>MSTN</i> biallelic mutations (left) were hypertrophic, compared to those of the wild-type calf (right). All animals were one month old on the date of the tissue sample collection.</p

Analysis of <i>MSTN</i> mRNA and N-terminal protein in mutant cell colonies.

<p>(A) RT-PCR analysis of mRNA transcribed from <i>MSTN</i> mutations in bovine fibroblasts. Among the biallelic mutant colonies, the MSTN mRNA was not expressed in colony 6, but was highly expressed in colony 7. Among the monoallelic mutant colonies, the expression of MSTN mRNA in colonies 20 and 44 was reduced. The WT <i>MSTN</i> allele (+) and/or mutant <i>MSTN</i> allele (–) are indicated at the top. <i>GAPDH</i> was used as a loading control. (B) Western blot analysis of <i>MSTN</i> mutants in bovine fibroblasts. The N-terminal MSTN was not expressed in colony 6, but was highly expressed in colony 7 and 20, compared with that in the WT cells. Total protein (50 µg) from bovine fibroblasts was subjected to SDS-PAGE on a 12% acrylamide gel, and the N-terminal MSTN was detected using a mouse anti-myostatin antibody. Latency-associated peptide (LAP) and the processed form of N-terminal MSTN are indicated. β-actin was used as a loading control.</p

Comparison of gene mutations induced using MSTN-ZFN mRNA (set 1) and plasmid DNA in single-cell colonies.

<p>*the mutant efficiency was calculated by mutant colonies/total single cell colonies.</p

MSTN mRNA sequencing and protein prediction in cloned cattle.

<p>(A) The 6-bp deletion in the splicing signal sequence in the <i>MSTN</i> ORF, which comprised one allele, resulted in three mRNA splice variants, in which a 22 nt deletion, a 291 nt deletion, or 97 nt insertion occurred, all of which caused the premature termination of translation (22 nt deletion, 97 nt insertion) or prevented translation completely (291 nt deletion). The 117-bp deletion and 9-bp insertion that comprised the other allele produced only one type of transcript, in which 2 consecutive nucleotide substitutions occurred, which resulted in one amino acid substitution (serine to arginine) in the MSTN protein. The red, blue, and green letters represent the sequences of exon 1, intron 1, and exon 2 of <i>MSTN</i>, respectively. The asterisk indicates the stop codon. (B) The frequencies of the mRNA splice variants. The mRNA frequency of the dinucleotide substitution was highest, followed by the 22-nt deletion, the 291-nt deletion, and the 97-nt insertion. The overall frequencies of the two alleles were equivalent.</p

The mutation efficiency of MSTN-ZFN (set 1) in different mammalian species.

<p>*The mutant efficiency is calculated by the mutant TA-cloning of PCR products of mixed cells/total sequencing number. The mismatch affected the ZFNs cut-efficiency dramatically. Lowercase letters were represented different nucleotides according to the targeting sequence of bovine. The bold italics represent the spacer nucleotides between the two ZFN monomers.</p

ELISA analysis of MSTN protein with a functional C-terminal domain in cloned cattle.

<p>We also produced a healthy cloned calf with monoallelic <i>MSTN</i> mutation, which consisted of a 55-bp insertion in intron 1, and caused the premature termination of translation. This allele is represented by “55 bp inserted” in the chart, and “6 bp/117 bp deleted” represents the double-muscled cloned bovine previously discussed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095225#pone-0095225-g004" target="_blank">Figure 4</a>. The level of MSTN protein with a functional C-terminal domain was reduced by approximately 50% in both mutant calves, compared to that of the WT calf. The ELISA data were analyzed using paired Student’s <i>t</i>-tests. The error bars represent the standard deviations of three experiments (*<i>P</i><0.05 indicates a statistically significant difference compared to the WT calf).</p

Use of ZFNs to disrupt bovine <i>MSTN</i>.

<p>(A) The target sequences of engineered ZFNs at the <i>MSTN</i> locus. The specific recognition sequences of the ZFNs included 19 bp upstream and 16 bp downstream of the <i>Fok</i>I non-specific cleavage sequence (AGTGT or ACACT). (B) Flowchart depicting the methodology used to generate ZFN-induce mutations in the single-cell colonies without drug selection. The mRNA was transcribed from the MSTN<i>-</i>ZFN plasmid in vitro, and the BFF cell line was derived from a Chinese domestic yellow cattle fetus. The BFF cells were transfected with MSTN-ZFN mRNA using the Neocleofector reagent for 24 to 48 h. Limiting dilution was used to form single-cell colonies at a cell concentration of approximately 500 cells/dish (10 cm²). The single-cell colonies were generated after culturing for an additional 6 to 7 days.</p