| Myostatin and follistatin have been proved as important regulators in the muscle development of mammals. Myostatin, a member of TGF-beta superfamilies, acts as a negative regulator during skeletal muscle growth. Mutations of myostatin in mice, cows, dogs and human led to muscular hypertrophy phenotype. Follistatin, as the antagonist of many TGF-beta members, participates in many physiological processes in different tissues, including the muscle. Overexpression of follistatin specifically in skeletal muscle leads to muscle hypertrophy, showing a similar phenotype to those of myostatin-deficient animals. Pig, as an indispensable meat source in food market, is one of significantly commercial animals. The economic value of pig in market mainly depends on quantity and quality of meat. Due to the enormous effects in muscle development, myostatin and follistatin could be the potential target genes for genetic modifications in agricultural animal breeding. In this study, we focused on the skeletal muscle of myostatin-knockout and muscle-specific follistatin transgenic pigs which were generated with traditional homologous recombination and transgene means on the basis of nuclear transfer technique, respectively.It was firstly identified with PCR and Southern blot that homologous recombination between endogeneous myostatin and targeting sequence was successful in FO pigs. Then a new transcript, which containing the first and second exons and part of second intron of myostatin, was characterized with RT-PCR and 3’RACE. The transcript was expected to encode a new protein which is similar to the N terminal propeptide of native myostatin. The C terminal of myostatin which acts as the functional domain was shown to be remarkably down-regulated at both the mRNA and protein levels according to the results of Q-PCR and ELISA in myostatin-knockout pigs. The phosphorylation of Smad2, which is the downstream and a media of myostatin signaling, displayed a lower level in the skeletal muscle of knockout pigs than that of wild-type controls. In addition, productive traits of knockout pigs from F1 generation were analyzed by ultrasonic detection and anatomy. The ultrasonic detection showed that lean percentage in heterozygous pigs was 1.04%(P=0.00536) higher than that in wild-type controls, and the backfat thickness in heterozygous pigs was 1.24% lower than that in wild-type pigs (P= 0.00771). While the measurement of anatomy showed that the lean percentage was 2.83% higher in pigs heterozygous than that in controls (P=0.00448), and the body fat percentage and leaf fat proportion in heterozygous pigs was 4.46% and 0.237% lower than that in controls (P=0.00024 and 0.00219, respectively). To determine the meat quality of knockout pigs, we conducted meat analysis using skeletal muscles. There was no significant difference in shearing force, intramuscular fat (IMF) percentage and pH of muscle between the knockout and wild-type pigs, while a significant difference in meat color was detected in heterozygous pigs.As for muscle-specific follistatin transgenic pig, fourteen transgene-positive pigs from three lines (TG-3, TG-6, TG-24) were identified by PCR and Southern blot. RT-PCR confirmed a muscle-specific expressional pattern of exogenous follistatin. As shown by ELISA, follistatin in three lines showed different expression levels in muscle. The follistatin expression in TG-6 and TG-24 lines were 49.21 pg/mg and 165.77 pg/mg, which are notably higher than that in wild-type controls (PTG-6 vs WT=0.0281, PTG-24 vs WT<0.0001). While the follistatin expression level in TG-3 line was not significantly different from that in controls (38.15 pg/mg vs 39.11 pg/mg, PTG-3 vs WT=0.8320). F1 generation of TG-24 line was produced, in which follistatin expression was the highest among three lines, for further study. The result from anatomy on F1 pigs at the age of 5.5-6 months showed 3.77%,2.043% and 0.816% higher in lean percentage, longissimus dorsi muscle proportion and semimembranosus muscle proportion of transgenic pigs than that of wild-type sibs, respectively (P=0.0118,0.0002 and 0.0006, respectively). While leaf fat proportion, skin and subcutaneous fat proportion in transgenic pigs were 1.563%,2.63% lower than that in wild-type sibs (P=0.0145 and 0.0264, respectively). To determine if skeletal muscle fibers in transgenic pig are hypertrophical, the myofiber diameters of transgenic pig and wild-type pig were analyzed. Compared with the myofiber of wild-type pig, the myofiber diameter of transgenic pig increased proportionally. Western blot showed down-regulated Smad2 phosphorylation and up-regulated Akt phosphorylation, suggesting that the increased follistatin expression inhibited TGF-P members and enhanced protein anabolism in muscle cells.The data of proteomic analysis also exhibited remarkably alternative signals involved inglycolysis/gluconeogenesis and biosynthesis of amino acids. Besides, meat qulity analysis showed no differences in digest yield, pH, IMF, water loss and shearing force between transgenic pigs and wild-type controls, except in meat color.In conclusion, our results from two genetically modified (GM) pigs (myostatin-knockout pig and muscle-specific follistatin pig) suggested myostatin and follistatin were functionally inhibited and improved in two GM pigs, respectively, both leading to enhanced skeletal muscle growth and declined fat accumulation. And our study supports the notion that myostatin and follistatin are the potential molecular targets for improving meat production, and provides experiences and scientific data for agricultural animal breeding using genetic engineering. |
