myostatin propeptide, follistatin, FLRG, monoclonal antibody for myostatin, activin receptor, Metalloproteases, myostatin receptor (name?), GASP-1 (Growth and Differentiation Factor-Associated Serum Protein-1)

follistatin-related gene (FLRG)

RNAi (RNA interference)- gene silencing methods?
* human oral RNAi isn't precisely working quite yet. (is it?)

myostatin antibodies would be the best, but there seems to be no available sequences for them, so either design a new antibody or go through the experiments to retrieve the antibodies (ELISA stuff I think)

follistatin
* needs some engineering so it does not bind to actin while maintaining myostatin-related activity



The function of myostatin and strategies for myostatin blockade


As mentioned above, the Myostatin propeptide can bind to Myostatin and thereby inhibiting Myostatin activity [6]. In two independent studies, Yang et al. [47] and Lee and McPherron [38] created transgenic mice that overexpressed the Myostatin propeptide under the control of the Myosin Light Chain (MLC) promotor. In the studies of Yang et al. [47] this resulted in an increase of carcass weight of 48% at 18 weeks. Morphometric analysis revealed an increase in fibre diameter but no increase in fibre number. Lee and McPherron [38] used the MLC promotor coupled to the 1/3 enhancer to drive the expression of the Myostatin propeptide region, which induced both muscle hypertrophy as well as hyperplasia.

In contrast to the transgenic approach, Wolfman et al. [45] injected protease resistant propeptide into adult mice, which resulted in an 18–27% weight increase of individual muscles within 4 weeks. Wolfman et al. [45] suggested that the mutated propeptide re-associates with mature Myostatin which then forms a complex that was resistant to the action of proteases. Interestingly, they showed that injection of wild type propeptide did not have any effect on muscle development, yet when this molecule was over-expressed in a transgenic approach it was able to promote muscle growth [38] and [47]. The reason for these apparent contradictory results are not clear, however, the transgenic approach might produce propeptide in such vast excess that it saturates protease activity.

[6] T.A. Zimmers, M.V. Davies and L.G. Koniaris et al., Induction of cachexia in mice by systemically administered Myostatin, Science 296 (2002), pp. 1486–1488. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (221)

[47] J. Yang, T. Ratovitski, J.P. Brady, M.B. Solomon, K.D. Wells and R.J. Wall, Expression of Myostatin pro domain results in muscular transgenic mice, Mol Reprod Dev 60 (2001), pp. 351–361. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (65)

Myostatin, also known as growth and differentiation factor 8 (GDF-8), belongs to the transforming growth factor beta (TGF-beta) superfamily. Mature TGF-beta proteins are generated by enzyme cleavage of the C-terminus of precursor molecules. The biologically active forms of TGF-beta are dimers, and remain associated with their N-terminal propeptides through noncovalent interactions in the forms of latent TGF-beta complexes (Gleizes et al., 1997). The biological activation mechanism from latent TGF-beta complexes to mature growth factors is not well understood. However, a mutation or truncation of the cleavage site has been demonstrated to be an effective way to block activities of TGF-beta family members (Gentry and Nash, 1990; Hawley et al., 1995). In transgenic mice, expression of a myostatin with a mutated cleavage site resulted in a decreased level of processed myostatin, and an increased level of uncleaved complex (Zhu et al., 2000). In COS-1 cell transient expression studies, the pro domain of TGF-beta 1 cDNA was able to form latent complexes with mature TGF-beta 1, inhibiting its biological activity (Gentry and Nash, 1990). Based on these obesrvations, we reasoned that over expression of the pro domain of myostatin would interfere with myostatin function to negatively regulate skeletal muscle development, thus promoting muscle growth.


Down-regulation of gene function by knock-out approaches is not yet established in swine. In order to down-regulate myostatin activity in pigs, transgene constructs might be designed to reduce myostatin RNA, block myostatin protein production, or interfere with myostatin function. Ribozyme-based transgenes are an effective way of reducing target RNA in tissue culture (Haseloff and Gerlach, 1988), but have a checkered record in transgenic animals. They have been successfully used in reduction of alpha-lactalbumin, glucokinase and beta-2-microglobulin mRNA in transgenic mice (Efrat et al., 1994; Larsson et al, 1994; L'Huillier et al.l, 1996). As a pilot study for a transgenic pig project, we have developed both ribozyme and myostatin pro domain approaches in an attempt to down-regulate myostatin activity. Expressing lines for both types of constructs were produced, but we detected no alteration in muscle growth in the ribozyme-carrying mice. However, the over-expression of myostatin pro domain DNA dramatically enhanced skeletal muscle development.

Ribozyme transgence. The secondary structure of mouse myostatin mRNA was predicted using software MFOLD version 3.1 (Zuker et al., 1999). Four potential target sites for hammerhead ribozymes (Perriman et al., 1992) were identified within regions of the mRNA sequence that did not appear to be constrained by secondary structure annealing. Hammerhead ribozymes were designed by inserting the consensus hammerhead motif (Haseloff and Gerlach, 1988) between flanking sequences complementary to each of the target sites in the mouse myostatin mRNA. Double stranded DNA fragments encoding the four individual ribozymes were created by annealing complementary synthetic oligonucleotides, and the individual ribozyme coding regions were ligated to one another to produce a multi-target ribozyme gene as descried by Chen et al. (1992) and Leopold et al. (1996). The multi-target ribozyme gene was flanked by inverted repeats so as to position the ribozymes sequences in the center of a stable stem-loop structure (Lieber and Strass, 1995).

Pro domain transgene. The coding sequence for myostatin pro domain was obstained by PCR amplification of the 5'-flanking 0.8 kb fragment of myostatin cDNA (Amino acid residue 1-266, Genbank accession number U84005). The T7 forward primer and reverse primer (5'-GCGGATCCTGAGCACCCACAGCG-3'), incorporating a Bam H I restriction site were used to generate GDF-8 cDNA for in-frame fusion with the FLAG epitope. The resulting cDNA was ligated into the pCR 2.1 vector (Invitrogene Carslbad CA) and sequenced for verification.

Transgene construction. Both ribozyme and myostatin pro domain DNA were inserted into the pMEX-NMCS2 vector, donated by Dr. Craig Neville at Massachusetts General Hospital (Rosenthal et al., 1990; Neville et al., 1996). pMEX-NNCS2 contains rat myosin light chain 1 (MLC1) regulatory sequences (1.5 Kb), SV40 splice/poly adenylation fragment (0.8 kb), and MLC enhancer (0.9 kb). The ribozyme construct was cloned into the Sal I/Hind III site of the vector pMEX-NNCS2. The 3.6 kb construct (MLC-Rib) was obtained by Not I digestion of the vector. The pro domain transgene was inserted into the EcoRI / Hind III site of pMEX-NMCS2 vector. The recombinant vector pMEX-NMCS2 was then digested with Not I restriction enzyme, resulting in the 4.2 kb construct (MLC-Pro, Fig. 1). Both transgene constructs includes MLC1 promoter, SV40 Poly adenylation sequence, and MLC enhancer in addition to the transgence sequence (ribozyme multimer or pro domain DNA sequence).

pronuclear microinjection techniques using B6SJL F1 females as zygote donors and CB6 females as embryo recipients. 

transgenic mice carrying a human growth hormone (HGH) transgene (Palmiter et al., 1982). Since then, the hormone cascade from growth hormone-releasing factor (GRF) and GH to insulin-like growth factor I (IGF-I) has been the target of transgenic projects designed to increase livestock growth and carcass quality (Pursel et al., 1989). In transgenic mice with GH from rat, human, and bovine, growth rates (5-11 weeks) were increased by four-folds. Transgenic pigs generated with a bGH transgene construct showed a significant increase in feed efficiency and a reduction in backfat thickness (Pursel et al., 1989, 1997). However, various animal health problems such as hepatomegaly, glomerular sclerosis, osteochondritis, and infertility in females were observed in those pigs. Transfers of GRF and c-ski transgenes have also been tested in livestock animals (Pursel et al., 1990; Bowen et al., 1994), but neither construct resulted in useful phenotypes. Recently, transgenic pigs carrying an IGF-I transgene have been generated. Lean tissue growth in the transgenic pigs was significantly improved with 10-20% larger loin eye area and lower body fat percentage in comparison with nontransgenic littermate controls (Pursel et al., 1999; Bee et al., 2001).

Mouse myostatin cDNA encodes a protein of 376 amino acids that is cleaved to produce an active protein of 109 amino acids, which negatively regulates both myoblast proliferation and fiber development (McPherron et al., 1997; Thomas et al., 2000). Directly targeting this kind of local effector with transgenic technology might offer more flexibility in controlling muscle development without adverse effects on other tissues. In consideration of potential applications of down-regulation of myostatin to improve muscle development in swine, we tested the feasibility of ribozyme and myostatin pro domain cDNA transgene constructs to depress myostatin activity in transgenic mice.

In transgenic mice expressing myostatin mutated at its cleavage site, levels of both myostatin RNA and protein were reduced (Zhu et al., 2000). We speculate that the amount of transgene mRNA we detected is likely correlated with the amount of myostatin pro domain protein in our transgenic mice. LIke the live weight, the muscular and carcass phenotypes of the HIGH expressing line were also intermediate between heterozygous and homozygous myostatin knock-out mice, where either 50% or no functional myostatin was present in the muscle. Therefore, functional myostatin is probably still present in the MLC-pro transgenic mice, and the muscular phenotype results from partially blocking myostatin biological activity.

[38] S.J. Lee and A.C. McPherron, Regulation of Myostatin activity and muscle growth, Proc Natl Acad Sci USA 98 (2001), pp. 9306–9311. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (303)

[45] N.M. Wolfman, A.C. McPherron and W.N. Pappano et al., Activation of latent Myostatin by the BMP-1/tolloid family of metalloproteinases, Proc Natl Acad Sci USA 100 (2003), pp. 15842–15846. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (93)

[61] L.A. Whittemore, K. Song and X. Li et al., Inhibition of Myostatin in adult mice increases skeletal muscle mass and strength, Biochem Biophys Res Commun 300 (2003), pp. 965–971. Article |  PDF (337 K)  | View Record in Scopus | Cited By in Scopus (123)