Electrophoresis methods

2.2.1 Agarose gel electrophoresis

Agarose gels were made to 1% agarose (w/v) in TAE buffer (40 mM Tris acetate, 1 mM EDTA). DNA samples were mixed at a 1:1 ratio with sucrose loading buffer (4% sucrose (w/v), 0.1% bromophenol blue) and gels were run at 100 V. Gels were stained in ethidium bromide (5 μg/mL in TAE) and visualized with the GelDoc XR gel documentation system and software (BioRad).

2.2.2 SDS-PAGE

SDS-PAGE was carried out according to Laemmli (Laemmli 1970). Samples were boiled in 5x SDS-PAGE sample buffer (0.125 M Tris-HCl pH 6.8, 35% glycerol, 2.5% SDS, 0.025% bromophenol blue) in the presence or absence of 2 M β- mercaptoethanol (βME) as a reducing agent and run at 150-200 V. All gels for visualisation of MstnPP were 12%; where observation of the myostatin growth factor was required, 15% gels were used. Gels were made in-house. Gels were stained with Coomassie Brilliant Blue R-250 (0.0625% w/v in 40% EtOH, 10% acetic acid),

destained in 20% EtOH, 10% acetic acid, and visualized using the GelDoc XR gel documentation system and software (BioRad).

2.2.3 Western blotting

Protein samples were separated on a 12% SDS-PAGE gel at 150 V and transferred to a nitrocellulose membrane overnight at 15 V (approximately 40-150 mA) in cooled Tris/glycine transfer buffer. For analysis of high molecular weight samples, transfer was continued the following day at 450 mA for one hour. Efficient transfer was confirmed by Ponceau S staining. Membranes were blocked with 5% non-fat milk in TBS-Tween (TBS-T; 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) for one hour at room temperature and subsequently incubated in primary antibody diluted in 1% non-fat milk TBS-T, for 90 minutes. Following washing with TBS-T (5 x 5 minutes), membranes were incubated in horse radish peroxidase-conjugated secondary antibody diluted in 1 % non-fat milk, TBS-T for 90 minutes. All steps were performed at room temperature. After washing with TBS-T (5 x 5 minutes), proteins were visualised with the SuperSignal West Pico Chemiluminescent Substrate (Pierce) according to manufacturer’s instructions.

Antibody concentrations depended on the detection method used. Details of antibodies and dilutions are summarised in Table 2.1.

2.3

Cloning

All myostatin constructs were produced from the full-length human myostatin cDNA (1-375) in the pBlueScript vector (from Se-Jin Lee, John Hopkins University School of Medicine, Baltimore, USA).

2.3.1 PCR

The myostatin precursor protein (MstnPP) construct was engineered to omit the N- terminal signal sequence. Because the published data does not agree with the site for the start of the propeptide region, three constructs were made initially, to begin at amino acids 19 (Bentsen, Nielsen et al. 2004), 21 (Jin, Dunn et al. 2004) and 25 (Lee 2004) (F19, F21 and F25 respectively). Forwards and reverse primers (Sigma- Genosys) contained BamHI and XhoI restriction sites (underlined below) respectively, for cloning into the pProxEX-Htb expression vector (Invitrogen, Appendix 2, Fig. 12.6). The forwards primers were as follows:

F19: ggt gga tcc ggt cca gtg gat cta aat gag F21: ggt gga tcc gtg gat cta aat gag aac agt F25: ggt gga tcc gag aac agt gag caa aaa gaa

The same reverse primer was used:

B375: ggt ctc gag tga gca ccc aca gcg gtc tac

PCR was carried out using 2.5 U of Roche PWO polymerase per 50 μL reaction. Each reaction contained 1 ng DNA template, 20 pmol of each primer and 0.5 mM dNTPs in a MgSO4-containing buffer. PCR incubation involved 35 cycles with denaturation at

95 °C, an annealing temperature of 58 °C and extension at 72 °C. PCR products were confirmed on agarose gel electrophoresis and purified using the Roche PCR purification kit.

2.3.2 Restriction digest

Restriction digests were performed on all cDNA constructs (10 μL per digest) and the pProEX-Htb vector (5 μL per digest), with a total digest volume of 20 μL. 1 U of

BamHI and XhoI (Roche) enzymes and 2 μL of 10 x Roche Buffer B were used, with digests carried out at 37 °C for one hour. cDNA digests were purified using the Roche

PCR purification kit. The entire vector reaction was electrophoresed on a 1% agarose gel and the vector backbone was excised under high-wavelength UV light, with purification by the Roche PCR purification kit, adapted for agarose gel products. Single- and no-enzyme digests were conducted as controls and analysed with agarose gel electrophoresis. DNA concentrations were established using NanoDrop technology (Thermo Scientific).

2.3.3 Ligation

A 3:1 molar ratio of insert to vector backbone was used per ligation. 1 U of T4 DNA ligase in T4 ligase ATP-buffer (Roche) was added in a total volume of 10 μL per reaction. A negative ligation control without insert was also performed. Ligations were incubated overnight at 4 °C.

2.3.4 Transformation

The entire ligation reaction was added to 50 μL competent Top 10 E.coli cells and placed on ice for 15 minutes. The incubations were heat-shocked at 42 °C for 1 minute and placed back on ice for 2 minutes before plating on LB-amp plates for overnight incubation at 37 °C.

2.3.5 Colony PCR

Colony PCR was used to determine recombinant colonies using pProEX forward and reverse sequencing primers. In brief, 9 colonies per plate were sampled by swirling a toothpick that had been touched to a colony in 25 μL buffer (50 mM Tris-HCl, pH 8.5, 150 mM NaCl). Toothpicks were subsequently used to scratch a grid on an LB- amp plate for growth of positive colonies. Solutions were boiled for 10 minutes then centrifuged at 17,000 x g, room temperature for 5 minutes and 5 μL from the bottom of each eppendorf was added to 20 μL PCR reaction mix containing 0.5 U Taq polymerase (Roche). Positive colonies were identified using agarose gel electrophoresis (Fig. 2.1) and used to inoculate overnight cultures for plasmid preparations (Roche Plasmid Purification kit).

Figure 2.1 Colony PCR of cells carrying pProEX-MstnF19/F21/F25 constructs.

Arrow indicates a positive result. A band just above 1.0 Kb represents myostatin precursor protein cDNA.

2.3.6 Sequencing

Traditional Sanger sequencing of all myostatin constructs was performed by The Allan Wilson Centre Genome Service, Massey University, Palmerston North, using ABI3730 capillary instruments. Primers were pProEX-Htb-specific forward and reverse sequencing primers. Sequences were viewed using 4Peaks software (Mac OS X) and confirmed by NCBI BLAST analysis.

2.3.7 Site-directed mutagenesis

The C313Y mutation was constructed using the splice-overlap-extension (SOE) method of site-directed mutagenesis (Fig. 2.2). Reverse complementary primers (Sigma-Genosys) containing the C313Y mutation (G938A by nucleic acid sequence) were designed and used in conjuction with the F21 and B375 forwards and reverse primers in two separate PCR reactions (Fig. 2.2a, blue and black arrows). The purified products, now containing the mutation (Roche PCR purification kit) were used in a second PCR reaction with F21 and B375 primers (Fig. 2.2b) to generate the full- length mutant construct (Fig. 2.2c). Following restriction digest, ligation, transformation and colony PCR, sequencing ensured the mutation had been created.

Figure 2.2 Splice-overlap extension (SOE) mutagenesis protocol.

a. Reverse complementary primers containing the Piedmontese mutation (red star) were used in conjunction with F21 (black arrow) and B375 (blue arrow) in two separate PCR reactions.

b. The purified products of a. were used in a second PCR reaction with F21 and B375. c. Purification of the PCR reaction product from b. generates the full-length mutant construct.

Mutagenesis primers are shown below; the mutation is highlighted in red: SDM_F tac tgc tct gga gag tat gaa ttt gta ttt

SDM_R aaa tac aaa ttc ata ctc cct aga gca gta