Biochemical and Biophysical Research Communications 477 (2016) 115e122 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc Establishment and phenotypic analysis of an Mstn knockout rat Hao Gu a, Yong Cao a, b, Bin Qiu a, Zhiqiang Zhou a, Ran Deng a, Zhuang Chen b, ** * Rongfeng Li c, Xueling Li c, Qiang Wei a, Xianzhu Xia d, , Weidong Yong a, a Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100021, China b Experimental Medicine Center, The First Affiliated Hospital of Sichuan Medical University, Sichuan 646000, China c The Key Laboratory of Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot 010021, China d Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130122, China article info abstract Article history: Myostatin (Mstn) is an inhibitor of myogenesis, regulating the number and size of skeletal myocytes. In Received 5 June 2016 addition to its myogenic regulatory function, Mstn plays important roles in the development of adipose Accepted 8 June 2016 tissues and in metabolism. In the present study, an Mstn knockout rat model was generated using the Available online 8 June 2016 zinc finger nuclease (ZFN) technique in order to further investigate the function and mechanism of Mstn in metabolism. The knockout possesses a frame shift mutation resulting in an early termination codon Keywords: and a truncated peptide of 109 amino acids rather than the full 376 amino acids. The absence of Mstn detectable mRNA confirmed successful knockout of Mstn. Relative to wild-type (WT) littermates, Knockout rats fi Muscle development Knockout (KO) rats exhibited signi cantly greater body weight, body circumference, and muscle mass. fi TGF-b However, no signi cant differences in grip force was observed, indicating that Mstn deletion results in greater muscle mass but not greater muscle fiber strength. Additionally, KO rats were found to possess less body fat relative to WT littermates, which is consistent with previous studies in mice and cattle. The aforementioned results indicate that Mstn knockout increases muscle mass while decreasing fat content, leading to observed increases in body weight and body circumference. The Mstn knockout rat model provides a novel means to study the role of Mstn in metabolism and Mstn-related muscle hypertrophy. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction function [7,8]. Mstn knockout mice exhibit dramatically increased muscle mass from an increased number of muscle fibers (hyper- Myostatin (Mstn), also known as growth and differentiation plasia) formed during development and increased fiber size (hy- factor 8 (GDF-8), is a member of the secreted transforming growth pertrophy) in adulthood [2,9]. Likewise, naturally occurring Mstn factor-b (TGF-b) superfamily of secreted growth factors and is a gene mutations generate the similar phenotype of muscular hy- negative regulator of skeletal muscle development [1]. In mam- pertrophy in many different mammalian species including cattle, mals, Mstn is primarily expressed in skeletal muscle [2], although sheep, dogs, and humans [10]. Mstn regulates muscle homeostasis, weak expression in other tissues such as adipose [2], heart [3], as myostatin upregulation is observed in skeletal muscle atrophy mammary gland [4], spleen [5], and placenta [6] have also been and in the pathogenesis of muscle wasting during cachexia asso- observed. The fact that the myostatin protein sequence is highly ciated with different diseases (i.e. cancer, chronic heart failure, conserved from rodents to human suggests conservation of AIDS, sarcopenia) [1,11]. What’s more, the use of antisense RNA, neutralizing antibodies, or chemical inhibitors against myostatin can improve this observed muscle wasting in animal models of e * Corresponding author. Institute of Laboratory Animal Science, Chinese Academy disease [1,12 17]. of Medical Sciences, Peking Union Medical College, Beijing 100021, China. The human Mstn gene is located on chromosome 2 (2q32.2) and ** Corresponding author. Key Laboratory of Jilin Province for Zoonosis Prevention is flanked by a number of binding sites and response elements for and Control, Institute of Military Veterinary, Academy of Military Medical Sciences, transcription factors and hormones, including MyoD (a myogenic Changchun 130122, China. regulatory factor), glucocorticoid, and androgen [18,19], suggesting E-mail addresses: [email protected] (X. Xia), [email protected] (W. Yong). http://dx.doi.org/10.1016/j.bbrc.2016.06.030 0006-291X/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 116 H. Gu et al. / Biochemical and Biophysical Research Communications 477 (2016) 115e122 myostatin could participate in multiple metabolic pathways. In 4. Methods addition to regulating muscle development and homeostasis, myostatin also exerts effects on adipocyte differentiation, as well as 4.1. ZFN construct design and in vitro transcription adipose accumulation and turnover. Myostatin has been shown to inhibit or stimulate adipogenesis in various cell culture systems ZFN constructs were co-designed by Sigma-Aldrich (MO, USA). [20], but the reduced expression of the adipogenic markers, such as The methods have been described in detail elsewhere [30e32].In CCAAT/enhancer binding protein a and peroxisome proliferator- brief, ZFNs were designed to target the genomic region around rat activated receptor g [21], as well as the reduced fat accumulation mstn exon 1 and each were tested for their efficiency in knocking in mstn-deficient mice [21e23], suggest myostatin promotes adi- out gene expression in the rat cell line C6. Cells were transfected pogenesis in vivo. Furthermore, mstn deficiency can prevent high- with the ZFNs and harvested the following day. DNA and RNA were fat diet-induced obesity through enhanced fatty acid oxidation isolated and Surveyor nuclease cleavage analysis was performed and brown adipose formation in white adipose tissue [24]. Finally, according to the manufacturer’s protocol (Transgenomic, NE, USA). myostatin has been found to reduce insulin sensitivity in response to neuronal stimulation of brown adipose tissue or via degradation 4.2. Creation of Mstn KO rat and sequencing confirmation of insulin receptor substrate 1 in response to high caloric intake [25,26]. Hence, myostatin appears to play an important role in Embryos and microinjection were performed as previously metabolic pathways not directly related to muscle growth. described [33].GenomicDNAfromtheZFN-modified founder animal Multiple studies have implicated myostatin as a potential target was sequenced to confirmthepresenceoftheZFN-inducedmutation. for many human diseases. Most of these studies were performed In brief, genomic DNA was extracted from the tails of newborn (10 d) using mouse models. Given the difficulty of modifying its largely rats using the salting-out method (ref). PCR was performed to amplify intractable genome, few studies were carried out in rat, another the Mstn sequence with the primers 50-GGCATGGTAAT- welleestablished model for the genetic dissection of human dis- GATTGTTTCCGTG-30 (forward) and 50-TTTACCTGTTTGTGCT- ease. From an evolutionary point of view, rats are more genetically GATTGCTGC-30 (reverse). Conditions for PCR amplification were set as: similar to humans and possess several genes not found in mice, 94 C pre-denaturation for 5 min, 94 C denaturation for 30 s, 60 C including genes involved in immunity, chemosensation, detoxifi- annealing for 30 s, 72 C elongation for 1 min, 35 cycles, and 72 C final cation and proteolysis. For proteolysis plays a regulatory function in elongation for 10 min. Confirmed ZFN-mediated Mstn knockout F1 myostatin signaling [27,28] and myostatin expression has an in- founder rats were backcrossed to WT Sprague Dawley rats. Hetero- fluence on cytokine secretion within the immune system [5], the zygous rats were crossed to generate Mstn homozygous KO rats. To activity of myostatin could be quite different between deficient rats reduce the probability of nonspecific ZFN-mediated deletion effects, F1 and deficient mice. In fact, Mendias et al. recently reported that the rats were backcrossed with WT Sprague Dawley rats for 5 generations. muscle and tendon phenotype of Mstn-null rats established using the zinc finger nuclease (ZFN) technique was markedly different 4.3. Mstn mutant genotyping from that of Mstn-null mice, which have impaired contractility and pathological changes to fibers and their extracellular matrix [29]. Genomic DNA was extracted from the tails newborn (10 d) rats. While that study demonstrated some important distinctions be- Primers and PCR conditions were employed as above. PCR products tween the mouse and rat models, it examined neither effects on fat were digested by ClaI restriction endonuclease at 30 C overnight content nor sex-specific phenotypes in Mstn-null rats. and fractioned on a 2% agarose gel. The present study characterizes a novel Mstn-knockout rat model developed using the ZFN technique, examining the muscle 4.4. RNA extraction and real-time PCR mass and fat contents, to provide a foundation for further studies of Mstn function. Rats