Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs

Abstract

Myostatin (MSTN) is a dominant inhibitor of skeletal muscle development and growth. Mutations in MSTN gene can lead to muscle hypertrophy or double-muscled (DM) phenotype in cattle, sheep, dog and human. However, there has not been reported significant muscle phenotypes in pigs in association with MSTN mutations. Pigs are an important source of meat production, as well as serve as a preferred animal model for the studies of human disease. To study the impacts of MSTN mutations on skeletal muscle growth in pigs, we generated MSTN-mutant Meishan pigs with no marker gene via zinc finger nucleases (ZFN) technology. The MSTN-mutant pigs developed and grew normally, had increased muscle mass with decreased fat accumulation compared with wild type pigs, and homozygote MSTN mutant (MSTN(-/-)) pigs had apparent DM phenotype, and individual muscle mass increased by 100% over their wild-type controls (MSTN(+/+)) at eight months of age as a result of myofiber hyperplasia. Interestingly, 20% MSTN-mutant pigs had one extra thoracic vertebra. The MSTN-mutant pigs will not only offer a way of fast genetic improvement of lean meat for local fat-type indigenous pig breeds, but also serve as an important large animal model for biomedical studies of musculoskeletal formation, development and diseases.

Figure 1. Target disruption of MSTN in Meishan (Ms) pigs via ZFNs and photos of MSTN mutant Ms pigs.

(A) Diagram of engineered ZFNs binding to MSTN exon 2. Red bar: ZFN-targeted site; gray box: exons; white box: introns. (B) Sequencing profiles of wide-type, hemizygous, and homozygous MSTN cell clones. Red box: ZFN-targeted site; dotted line: base deletions in homozygous MSTN modification. Please note that there are double peaks next to or near the ZFN cleavage site in heterozygous MSTN cell clone (see blue box). (C) Distribution of different MSTN mutation types. Of the 80 mutant cell colonies analyzed, the most dominant mutation types are short-fragment (1–10 bp) deletions. Vertical axis represents the percentage of each mutant type. Horizontal axis represents the sizes of ZFN-mediated fragment deletion (−) and insertion (+). (D) Sequences of ZFN-mediated disruption of MSTN gene in mutant cell lines. Deletions and insertions are indicated by red dashes and red letters, respectively. Sizes of base insertions (+) or base deletions (Δ) are indicated to the right of each mutant allele. Cell colony #172 is a MSTN homozygous mutant that contains deletion of one basepair. However, there is a possibility that alleles with a large deletion might not be detected. **Indicates that cloned live piglets were produced from these cell lines. (E) Photos of representative of MSTN^−/−, MSTN^+/− and MSTN^+/+ pigs (4-month old) generated via breeding F1 heterozygous pigs. Note that MSTN^−/− pigs show wider back, fuller rump and thicker limbs compared with MSTN^+/− and MSTN^+/+ pigs.

Figure 2. Changes of MSTN mRNA and protein in MSTN mutant Ms pigs.

(A) Agarose gel electrophoresis of RT-PCR products derived from the coding sequence of MSTN mRNA. A single band was obtained in MSTN^+/+ and MSTN^−/− pigs respectively, but there is approximate 200 bp difference in size between these two bands. Two bands were obtained MSTN^+/− pigs. (B) Change in splicing of MSTN mRNA exons after a T-G mutation. (C) Sequencing of the RT-PCR products indicated that MSTN gene from MSTN^−/− pigs had a T-G mutation at the beginning of intron 2 except for a 15-bp deletion, which result in intron error splicing—the 3’end of exon 2, resulting in a 193-nt deletion. The altered splicing of RNA caused frameshift, resulting in a premature termination of translation. Red, blue, and green letters represent the sequences of exon 2, intron 2, and exon 3 of MSTN gene respectively. Green underline: ZFN-targeted site; red box: single nucleotide mutation; and asterisk indicates the stop codon. (D) Real time quantitative PCR results of MSTN. Total RNAs were isolated from longissimus dorsi of MSTN^+/+, MSTN^+/− and MSTN^−/− pigs. The expression levels were analyzed using the ΔΔCt method and normalized against GAPDH. Each sample was run in triplicate. (E) Detection of MSTN protein in skeletal muscle (longissimus dorsi) by Western blot. Protein extracts (20 μg) from of 8-month-old male pigs were subjected to SDS-polyacrylamide gel electrophoresis, blotted and probed with anti-MSTN antibody. Precursor (52 kDa) and mature dimer (26 kDa) were indicated by arrow. GAPDH protein was used as an internal reference to demonstrate equal amounts of proteins were loaded. (F) ELISA analysis of MSTN protein in porcine serum. Mature MSTN protein wasn’t detected in MSTN^−/− pigs using an antibody recognizing the C-terminal domain of MSTN protein, and the level of MSTN protein in MSTN^+/− serum decreased compared with MSTN^+/+ serum. The error bars represent the standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test.

Figure 3. The increased body weight results from an increase in muscle mass in of MSTN mutant Ms pigs.

(A) Changes in average body weight of MSTN^+/+, MSTN^+/− and MSTN^−/− pigs from F2 sib or half-sib families at different ages (n = 3–6). (B) Average carcass weight of F2 8-month-old male pigs (n = 3–6). (C) Relative percentage of lean (a), fat (b), skeleton (c) and skin (d) of carcass weight in 8-month-old male pigs. (D) Average weight of individual skeletal muscles which were dissected on one side of the body. Black bar: MSTN^+/+; blue bar: MSTN^+/−; red bar: MSTN^−/−. Samples were collected from 8-month-old male pigs. Data are expressed as mean  ±  SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test.

Figure 4. Increased muscle mass of MSTN^−/− Meishan (Ms) pigs is a result of hyperplasia rather than hypertrophy of muscle fibers.

(A) Changes in myofiber size and density in MSTN editing Ms pigs. Histological cross section of longissimus dorsi (a) and semitendinosus (b). Scale bar = 50 μm. Average size and density of myofiber in longissimus dorsi (c,d) and semitendinosus (e,f). Distribution of different sizes of myofibers in longissimus dorsi (g) and semitendinosus (h) from MSTN^+/+, MSTN^+/− and MSTN^−/− pigs. (B) Loin eye area and number of myofiber in longissimus dorsi. Photos of cross section of longissimus dorsi (called loin eye muscle) at the last rib (a–c); loin eye area of longissimus dorsi from MSTN^+/+, MSTN^+/− and MSTN^−/− pigs (d); and relative number of myofiber in longissimus dorsi (LD) calculated from loin eye area and myofiber density (e). Black bar: MSTN^+/+; blue bar: MSTN^+/−; red bar: MSTN^−/−. Samples were collected from 8-month-old male pigs. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test.

Figure 5. Skeletal patterns in wild-type and MSTN mutant Meishan (Ms) pigs.

An example of one MSTN mutant pig (right) that contains fifteen thoracic vertebrae and one WT pig (left) that commonly contains fourteen thoracic vertebrae.