IMPROVING SKELETAL MUSCLE GROWTH OF FARM ANIMALS THROUGH MYOSTATIN-BASED BIOTECHNOLOGIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0233420
• Project Start Date: Apr 1, 2013
• Project End Date: Sep 30, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822

Performing Department
Human Nutrition, Food & Animal Sciences

Non Technical Summary
Meat animal producers in Hawaii need to improve production efficiency to prosper in the increasingly competitive market and to satisfy consumer demands for high quality meat and meat products at an economic price. Since the feed energy used for fat deposition is wasteful in meat animal production, enhancing skeletal muscle growth and suppressing carcass fat deposition will greatly improve the production efficiency of meat animals. It is, thus, imperative to develop strategies of improving skeletal muscle growth. One strategy of improving skeletal muscle growth is to identify target molecules that are critical in regulating the growth process of skeletal muscles, then modulate the biological activity of the target molecules in a way to maximize the skeletal muscle growth. Recent studies have unequivocally shown that myostatin (MSTN) is the most significant negative regulator of skeletal muscle growth in animals. Unlike other growth factors or hormones regulating animal growth, MSTN demonstrates exquisite specificity of its action on skeletal muscle, making it an ideal target molecule in modulating skeletal muscle growth. Various MSTN-suppressing molecules have been identified, and these molecules have been shown their potential to improve muscle growth in laboratory animals. However, the potential has not been examined in meat producing animals. To fill this gap, this project is designed (1) to optimize the expression and purification of various MSTN-suppressing molecules (MSTN-pro, soluble forms of ActRIIB-ECD, and follistatin) of farm animal species, 2) to examine the MSTN-suppressing capacity of those proteins in vitro and in vivo systems. These objectives address the CSREES goal, "enhance economic opportunities for agricultural producers."

Animal Health Component

30%

Research Effort Categories

Basic

30%

Applied

30%

Developmental

40%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3220	1020	15%
308	3220	1040	15%
305	3520	1020	15%
308	3520	1040	15%
305	3840	1020	20%
308	3840	1040	20%

Knowledge Area
305 - Animal Physiological Processes; 308 - Improved Animal Products (Before Harvest);

Subject Of Investigation
3220 - Meat-type chicken, live animal; 3840 - Laboratory animals; 3520 - Meat, swine;

Field Of Science
1040 - Molecular biology; 1020 - Physiology;

Keywords

skeletal muscle growth

biotechnology

myostatin

myostatin-based biotechnology

meat-producing animal

Goals / Objectives
The long term goal of this project is to enhance skeletal muscle growth of meat-producing animals by modulating the biological activity of myostatin (MSTN) through the utilization various MSTN binding proteins, including MSTN-pro, soluble forms of ActRIIB-ECD, and follistatin. The specific objectives of the proposal are 1)To optimize the expression and purification of various MSTN-suppressing molecules (MSTN-pro, soluble forms of ActRIIB-ECD, and follistatin) of farm animal species, 2) To examine the MSTN-suppressing capacity of those proteins in vitro and in vivo systems. Expected outputs are as follows: Knowledge on the optimum conditions of recombinant protein expression, Expression of large quantity (gram level) of recombinant proteins, Knowledge on the optimum purification conditions of expressed recombinant proteins, Purification of large quantity (gram level) of recombinant proteins, Knowledge on bioactivity of biochemical characteristics of the recombinant proteins, Knowledge on the effect of the administration of the recombinant proteins in chicken skeletal muscle growth, Knowledge on mechanistic responses in skeletal muscles in association with myostatin inhibition through the administration of myostatin-inhibiting molecules, Clear insight into the potentials of the administration of myostatin-inhibiting molecules in farm animal production, At least two or three refereed publication reporting the findings, One or two master or PhD student trained in molecular life science area.

Project Methods
1. Optimization of expression and purification of MSTN-binding proteins: Various strains of E. coli will be examined for their ability to express soluble forms of the recombinant proteins at different induction temperatures. Plasmids of the constructed vectors will be used to transform 6 or 7 different E. coli strains. The transformants will be spread on Luria-Bertani (LB) agar plates containing X-gal and appropriate antibiotics. Selected colonies will be used to inoculate 50 mL LB medium containing appropriate antibiotics, and cells will be grown to an OD600 of 0.4-0.5. To induce the expression of soluble recombinant MSTN prodomain,IPTG (0.2 mM final concentration) will be added to the cultures , then the cultures will be grown at different temperatures for 24 hrs. Thereafter, E. coli cells will be harvested by centrifugation. Pellets will be resuspended in an affinity buffer, and disrupted by sonication. Soluble and insoluble fractions will be separated by centrifugation. Total, soluble and insoluble fractions will be subjected to SDS-PAGE analysis for the examination of soluble expression of recombinant proteins. To determine the optimum induction time, samples will be collected at various times after IPTG induction. Amylose affinity chromatography will be used to purify the soluble recombinant proteins. Fractions containing the target protein will be pooled and dialyzed in 20 mM Tris-HCl buffer (pH 8.0), then stored at -20 C for later use in biological activity testing in vitro. Other chromatographic materials such as cationic starch will be tested for their efficacy of purification along with the combination of ion-exchange chromatographic steps. 2. Examination of the MSTN-suppressing capacity of MSTN-binding proteins in vitro and in vivo systems: Many laboratories have been using the pGL3-(CAGA)12 Luciferase assay system to measure the biological activity of MSTN. The PI laboratory has also established and tested the robustness of the pGL3-(CAGA)12 luciferase assay system as a measure of the MSTN activity. This reporter gene assay system will be used as a preliminary screening of MSTN-inhibitory capacity of the recombinant proteins. After confirming the ability of the recombinant proteins to suppress MSTN in in vitro reporter gene assay system, we will select one or two candidate proteins and move on to in vivo administration study. Since plasma clearance of the injected proteins will be an important factor affecting the effectiveness of the proteins to inhibit MSTN activity, obtaining information about the half-life of the injected proteins will be useful in gaining insights into the dose and frequency of injection. We will employ chicken as a farm animal model in the proposed study.

Progress 04/01/13 to 09/30/18

Outputs
Target Audience:Academics and industrial researchers and students, who are interested in improving the efficiency of meat animal production, as well as improving meat quality characteristics. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student had an opportunity to be trained in the biotechnology field, including gene cloning for recombinant protein production, protein purification, examination of bioactivity using cell-based reporter gene assay, and lab animal handling and sample collection. How have the results been disseminated to communities of interest?The results were presented in a college-sponsored conference. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Bioactive, recombinant myostatin propeptide conjugated to Fc domain of mouse IgG (MSTNpro-Fc) was proudced in E. coli and purified by doulbe affinity chromatography. Effects of MSTNpro-Fc on the growth and muscle mass functionality of adult mice was examined. The administration of MSTNpro-Fc for 5 weeks had no significant effect on body and muscle weights, but significantly enhanced the grip strength, suggesting an improved muscle functionality by the administration of the recombinant MSTNpro-Fc. Results of this study demonstrate the potential of the MSTNNpro-Fc to ehnhance muscle functionality in adult animals.

Publications

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Academics and industrial researchers and students, who are interested in improving the efficiency of meat animal production, as well as improving meat quality characteristics. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student has an opportunity to be trained in biotechnology field, including recombinant protein production and purification, cell-based reporter gene assay, and skeletal muscle cell culture. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?The MSTNpro will be examined for its potential to improve skeletal muscle growth in model animals.

Impacts
What was accomplished under these goals? A MSTN-inhibitory protein, Myostatin propeptide (MSTNpro), was produced in an E. coli system. The purified MSTNpro showed strong MSTN inhibitory capacity in cell-based assay system. When its effect on muscle cell proliferation was examined using C2C12 cell culture, the MSTN pro significantly enhanced cell proliferation, demonstrating the potential of MSTNpro to enhance skeletal muscle cell proliferation.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: J.D. Berrocoso, R. Kilda, A.K. Singh, Y.S. Kim and R. Jha. 2017. Effect of in ovo injection of raffinose on growth performance and gut health parameters of broiler chicken. Poultry Science 96:1573-1580.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: J-D. Kang, S. Kim, H-Y. Zhu, L. Jin, Q. Guo, X-C. Li, Y-C. Zhang, X-X. Xing, M-F. Xuan, G-L. Zhang, Q-R. Luo, Y.S. Kim, C-D. Cui, W-X.Li, Z-Y. Cui, J-S. Kim, and X-J. Yin. 2017. Generation of cloned adult muscular pigs with myostatin gene mutation by genetic engineering. RSC Adv., 2017,7, 12541-12549 DOI: 10.1039/C6RA28579A

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Academic and industrial researchers and students, who are interested in improving the efficiency of meat animal production, as well as improving meat quality characteristics. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student had an opportunity to be trained in advanced skills related to agricultural biotechnology. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We plan to examine biological effects of the myostatin-inhibitors in animal models.

Impacts
What was accomplished under these goals? Four different truncated forms of pig MSTNpro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in E. coli, and purified by the combination of affinity chromatography and gel filtration. The MSTN-inhibitory capacities of these proteins were examined in an in vitro gene reporter assay. This study showed that MBP is a very useful fusion partner in enhancing MSTN-inhibitory potency of truncated forms of MSTNpro proteins, and MBP-fused pig MSTNpro consisting of amino acid residues 42-175 is sufficient to maintain the full MSTN-inhibitory capacity.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Putluru, R.K., Kim, Y.S., and Lee, C.N. 2016. Differential expression of superoxide mutases (SODs) in bovine corpus luteum during estrous cycle and pregnancy. Pacific Agriculture and Natural Resources. Pacific Agticulture and Natural Resources, Vol. 1:11-20.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Kim, Y.S., Fukumoto, G., Stevenson, M., Thorne, M., and Jha, R. 2016. Carcass traits and tenderness of grass-fed beef from subtropical pastures in Hawaii. 17th Asian Australian Animal Production Animal Science Congress. August 22-25, 2016, Fukuoka, Japan.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Fukumoto, G.K., Kim, Y.S., and Kealoha, P. 2016. Evaluation of incorporating an improved leucanea forage for grass-fed beef production in a tropical ecosystem. 17th Asian Australian Animal Production Animal Science Congress. August 22-25, 2016, Fukuoka, Japan.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Berrocoso, J.D., Kida, R., Singh, A.K., Kim, Y.S., and Jha, R. 2016. In-ovo inoculation of raffinose (as a prebiotic) improves hatchability rate and gut health, but not growth performance of broiler chickens. The XXV Worlds Poultry Congress. September 5-9, 2016, Beijing, China.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audiences are academic and industrial researchers and students, who are interested in improving animal growth efficiency through myostatin-based biotechnology, as well as who are interested in improving meat quality. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Conference presentations and invited presentations. What do you plan to do during the next reporting period to accomplish the goals?We plan to examine the bio-stability and safety in animal models.

Impacts
What was accomplished under these goals? We are currently in the process of producing various recombinant myostatin-suppressing proteins, and some proteins have been tested for their myostatin-inhibitory capacities. Results of the study have been presented in scientific conferences.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Park, S.K., Jeong, Y.D., Lee, S.B., and Kim, Y.S. 2015. Porcine myostatin propeptide promotes the proliferation of satellite cells isolated from porcine skeletal muscle. International Conference of the Korean Society for Molecular and Cellular Biology. Sep 21-23. Seoul, Korea.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kim, Y.S., Lee, S.B., Park, S.K., and Jin, H-J. 2015. Production of bioactive myostatin propeptide of various animal species in E. coli. 7th Asia-Pacific Biotech Congress. J. Biotechnol. Biomater Vol 5, Issue 2:29

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The target audiences are academic and industrial researchers and students, who are interested in improving animal growth efficiency through myostatin-based biotechnology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A graduate student participated in an international conference to present findings of his study. How have the results been disseminated to communities of interest? Peer-reviewed publication and conference presentations. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We are currently in the process of producing various recombinant myostatin-suppressing proteins.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: D. Choi, J. Yang, S.K. Park, and Y.S. Kim. 2014. Muscle hypertrophy induced by myostatin inhibition is suppressed by rapamysin administration. J. Anim. Sci. 92 (suppl. 2): 588.

Progress 04/01/13 to 09/30/13

Outputs
Target Audience: The target audiences are academic and industrial researchers and students, who are interested in improving animal growth efficiency through myostatin-based biotechnology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A senior undergraduate student had conduced a series of experiments in relation to this project under the supervision of the PI, and submitted honor's program thesis using the data collected from the research. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We are currently in the process of producing various recombinant myostatin-suppressing proteins.

Publications