Lili Qian

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

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.

Loss-of-function myostatin mutation increases insulin sensitivity and browning of white fat in Meishan pigs

Myostatin-deficient mice showed a remarkable hypertrophy of skeletal muscle, with a decreased fat mass and enhanced insulin sensitivity. Currently, it is unclear if the inhibition of myostatin could be used as an approach to treat human obesity and insulin resistance. In this study, we investigated if the inhibition of porcine myostatin has any effect on fat deposition and insulin sensitivity using genetically engineered Meishan pigs containing a myostatin loss-of-function mutation (Mstn −/− ). Our results indicated that, when compared with wild-type pigs, the amount of subcutaneous fat and leaf fat of Mstn −/− pigs were significantly decreased mainly due to the browning of subcutaneous adipose tissue. Additionally, the serum insulin level decreased and the insulin sensitivity increased significantly in Mstn −/− pigs. Moreover, we found a significant increase in levels of insulin receptor and insulin receptor substrate proteins in skeletal muscle of Mstn −/− pigs, which then activating the insulin signaling pathway. Irisin-mediated regulation is not the only pathway for the activation of insulin signal in Mstn −/− skeletal muscle. This study provides valuable insight for the treatment of human obesity and diabetes mellitus.

Over-Expression of Porcine Myostatin Missense Mutant Leads to A Gender Difference in Skeletal Muscle Growth between Transgenic Male and Female Mice

Myostatin, a transforming growth factor-β family member, is a negative regulator of skeletal muscle development and growth. Piedmontese cattle breeds have a missense mutation, which results in a cysteine to tyrosine substitution in the mature myostatin protein (C313Y). This loss-of-function mutation in myostatin results in a double-muscled phenotype in cattle. Myostatin propeptide is an inhibitor of myostatin activity and is considered a potential agent to stimulate muscle growth in livestock. In this study, we generated transgenic mice overexpressing porcine myostatin missense mutant (pmMS), C313Y, and wild-type porcine myostatin propeptide (ppMS), respectively, to examine their effects on muscle growth in mice. Enhanced muscle growth was observed in both pmMS and ppMS transgenic female mice and also in ppMS transgenic male mice. However, there was no enhanced muscle growth observed in pmMS transgenic male mice. To explore why there is such a big difference in muscle growth between pmMS and ppMS transgenic male mice, the expression level of androgen receptor (AR) mutant AR45 was measured by Western blot. Results indicated that AR45 expression significantly increased in pmMS transgenic male mice while it decreased dramatically in ppMS transgenic male mice. Our data demonstrate that both pmMS and ppMS act as myostatin inhibitors in the regulation of muscle growth, but the effect of pmMS in male mice is reversed by an increased AR45 expression. These results provide useful insight and basic theory to future studies on improving pork quality by genetically manipulating myostatin expression or by regulating myostatin activity.

Functional and safety evaluation of transgenic pork rich in omega-3 fatty acids

Genetically modified animals rich in omega-3 unsaturated fatty acid offer a new strategy to improve the human health, but at the same time present a challenge in terms of food safety assessment. In this study, we evaluated the function and safety of sFat-1 transgenic pork rich in omega-3 fatty acids in mice by feeding basic diet and diets that contain wild type pork and sFat-1 transgenic pork. Blood biochemistry, haematology, peripheral T cell distributions, bacterial counts, gross necropsy, histopathology and organ weights were performed in mice fed with different doses of wild type and transgenic pork. Results indicated that both low and high dose of wild type and transgenic pork had no significant effect on blood biochemistry, T cell distribution, immunoglobulins and bacterial counts in intestine and feces. However, it was noted that both low and high dose of transgenic pork improved the liver immune system in mice, which is probably due to the beneficial contribution of high level of the “good” fatty acids in transgenic pork. There is no significant effect of transgenic pork on all other organs in mice. In summary, our study clearly demonstrated that feeding transgenic pork rich in omega-3 fatty acids did not cause any harm to mice, and in fact, improved the liver immune system.

Safety Evaluation of Neo Transgenic Pigs by Studying Changes in Gut Microbiota Using High-Throughput Sequencing Technology

The neo (neomycin phosphotransferase) gene is widely used as a selection marker in the production of genetically engineered animals and plants. Recent attention has been focused on safety concerns regarding neo transgene expression. In this study, neo transgenic and non-transgenic piglets were randomly assigned into Group A and Group B to evaluate effects of neo transgene by studying changes in gut microbiota using high-throughput sequencing. Group A pigs were fed a standard diet supplemented with antibiotic neomycin; Group B pigs were fed a standard diet. We examined horizontal transfer of exogenous neo gene using multiplex PCR; and investigated if the presence of secreted NPT II (neo expression product) in the intestine could lead to some protection against neomycin in transgenic pigs by monitoring different patterns of changes in gut microbiota in Group A animals. The unintended effects of neo transgene on gut microbiota were studied in Group B animals. Horizontal gene transfer was not detected in gut microbiota of any transgenic pigs. In Group A, a significant difference was observed between transgenic pigs and non-transgenic pigs in pattern of changes in Proteobacteria populations in fecal samples during and post neomycin feeding. In Group B, there were significant differences in the relative abundance of phyla Firmicutes, Bacteroidetes and Proteobacteria, and genera Lactobacillus and Escherichia-Shigella-Hafnia between transgenic pigs and non-transgenic pigs. We speculate that the secretion of NPT II from transgenic tissues/cells into gut microbiota results in the inhibition of neomycin activity and the different patterns of changes in bacterial populations. Furthermore, the neo gene also leads to unintended effects on gut microbiota in transgenic pigs that were fed with basic diet (not supplemented with neomycin). Thus, our data in this study caution that wide use of the neo transgene in genetically engineered animals should be carefully considered and fully assessed.

Gene Location, Expression, and Function of FNDC5 in Meishan Pigs

Irisin is a new muscular regulatory factor that is generated by the cleavage of its precursor protein fibronectin type III domain-containing protein 5 (FNDC5). Irisin promotes fat consumption due to its stimulatory role in the browning of the adipocytes in mice. Currently, there is no report on FNDC5 functions in pigs as model animals. In this study, we investigated the expression patterns and functions of FNDC5 in Meishan pigs. Our results showed that FNDC5 gene in Meishan pigs contains five transcripts, all of which can be translated into functional intact irisin proteins. Porcine FNDC5 is mainly expressed in skeletal muscle, with the expression level being significantly higher during the embryonic and juvenile periods than in the adulthood stage. In vitro study showed that FNDC5 stimulates the proliferation and adipogenic differentiation of primary adipocytes isolated from Meishan pigs, and FNDC5 enhances the expression of browning marker genes during adipogenic differentiation. Our study was the first report on FNDC5 expression patterns and functions in pigs. Data from this study provide valuable information related to the study on FNDC5 functions and future development of novel treatment for obesity.

MicroRNA-95 promotes myogenic differentiation by down-regulation of aminoacyl-tRNA synthase complex-interacting multifunctional protein 2

MicroRNA-95 (miR-95) is well known for its ability to promote the proliferation of a variety of cancer cells, but its function in skeletal muscle development has not been reported so far. Our laboratory has recently generated genetically engineered Meishan pigs containing a loss-of-function myostatin (MSTN) mutant (MSTN-/-). These MSTN-/- pigs grow and develop normally but show clear double muscle phenotype as observed in Belgian cattle. We observed that the expression of miR-95 was up-regulated in the longissimus dorsi from MSTN-/- Meishan pigs at day 65 during embryo development. In this study, we investigated the role of miR-95 in the myogenic differentiation using a murine myoblast cell line C2C12. Our results revealed that miR-95 may play a very important role in regulating the expression of myogenic differentiation marker genes myosin heavy chain (MHC) and myogenin. By use of bioinformatical analysis and luciferase reporter gene assay, aminoacyl-tRNA synthase complex-interacting multifunctional protein 2 (AIMP2) gene was identified as a miR-95 target gene involved in myogenic differentiation. Our results indicated that higher miR-95 expression level leads to lower level of AIMP2 protein expression. When the endogenous expression of AIMP2 is inhibited by siRNA, the expression levels of myogenic differentiation marker genes MHC and myogenin increased, implying that AIMP2 negatively regulates myogenic differentiation. Taken together, it is likely that miR-95 promotes myogenic differentiation in C2C12 myoblasts and may play a positive functional role in skeletal muscle development by down regulating the expression of AIMP2 at protein level.

A 90-Day Feeding Study in Rats to Assess the Safety of Genetically Engineered Pork

Our laboratory recently produced genetically engineered (GE) Meishan pigs containing a ZFN-edited myostatin loss-of-function mutant. These GE pigs develop and grow as normal as wild type pigs but produce pork with greater lean yield and lower fat mass. To assess any potential subchronic toxicity risks of this GE pork, a 90-day feeding study was conducted in Sprague-Dawley rats. Rats were randomly divided into five groups, and fed for 90 days with basic diet and basic diets formulated with low dose and high dose pork prepared from wild type pigs and GE pigs, respectively. Animal behaviors and clinical signs were monitored twice daily, and body weight and food consumption were measured and recorded weekly. At days 45 and 90, blood tests (lipid panel, electrolytes, parameters related to liver and kidney functions, and complete blood counts) were performed. Additionally, gross pathology and histopathological analyses were performed for major organs in each group. Data analysis shows that there were no significant differences in growth rate, food consumption, and blood test parameters between rat groups fed with GE pork and wild type pork. Although differences in some liver function parameters (such as aspartate aminotransferase, total proteins, albumin, and alkaline phosphatase) and white blood cell counts (such as lymphocyte percentage and monocyte percentage) were observed between rats fed with high dose GE pork and basic diet, all test results in rats fed with GE pork are in the normal range. Additionally, there are no apparent lesions noted in all organs isolated from rats in all five feeding groups on days 45 and 90. Overall, our results clearly indicate that food consumption of GE pork produced by ZFN-edited myostatin loss-of-function mutant pigs did not have any long-term adverse effects on the health status in rats.

Muscle hypertrophy in transgenic mice due to over-expression of porcine myostatin mutated at its cleavage site

Abstract Myostatin, a member of the transforming growth factor beta (TGF-β) superfamily, is a dominant inhibitor that acts to limit skeletal muscle growth and development. In this study, we generated transgenic mice that express porcine myostatin containg mutations at its cleavage site (RSRR) to evaluate its effect on muscle mass. Results showed that the weight of four skeletal muscles including gastrocnemius, rectus femoris, tibialis anterior, and pectoralis increased by 17.83 and 28.39%, 21.76 and 28.70%, 34.31 and 41.62%, 53.21 and 27.54% in transgenic male and female mice, respectively, compared to their corresponding non-transgenic control mice. Measurement of muscle fiber size and number indicated that the mean myofiber size increased by 50.73 and 61.30% in transgenic male and female mice respectively compared to the non-transgenic controls. However, there was no difference in the number of myofiber between transgenic and non-transgenic male mice. These results clearly demonstrated that the increase in skeletal muscle mass in transgenic mice is caused by hypertrophy instead of hyperplasia.