Primer design and oligo synthesis
Three primers were used for ABI-REC cloning. Primer pF(1+2) was composed of two parts: The P2 primer was complementary to the insert and the P1 primer was identical to the 5’ sequence of pre-selected insertion site. Primer pR(3+4) consisted of two arms: the P4 arm was complementary to the insert and P3 arm annealed to the 3’ sequence of insertion site. Primer P1R was complementary to primer P1. The annealing temperature in the PCR reaction was 5°C less than the Tm of primer P1R. The sequences of all oligos used in this work were chemically synthesized by Invitrogen (Shanghai) and are listed in Table . Note that oligos longer than 40bp were PAGE-purified, whereas those shorter than 40bp were desalted. All oligos were dissolved in TE (pH8.0) buffer and stored at -20°C.
Primers and oligos used in this study
Bacterial strains, backbone plasmids and DNA templates
E.coli strain DH5α [genotype:F- supE44_lacU169 f80 lacZ_ M15 hsdR17 recA1 0endA1 gyrA96 thi-1 relA1] (Takara, Japan) was used for all DNA cloning assays in this study. It was grown in LB medium (Luria-Bertani), where ampicillin and kanamycin were added at 50μg/ml and 100μg/ml, respectively, when necessary. Exogenous DNA was introduced into competent DH5α cells by chemical transformation. The pUC19 cloning plasmid was from Takara (Japan). pIRES2-EGFP was purchased from Clontech (U.S.). PGL3-basic, pGL3-promoter and pRL-TK luciferase reporters were purchased from Promega (U.S.). All these plasmids were cultured and propagated according to the manufacturers’ specifications and purified using an Ultrapure Plasmid Extraction Kit (Tiangen, Beijing, China). Hubei white pig ear tissue genomic DNA was extracted using a Roche High Pure PCR Template Preparation Kit. All DNAs solutions were measured using a NanoVueTM (GE Healthcare) spectrophotometer to determine purity and concentration.
Enzymes and reagents
ApaI, SalI, DpnI, BglII and XbaI restriction nucleases, Taq DNA polymerase and dNTP were purchased from Fermentas (Lithuania). KOD Plus high-fidelity DNA polymerase was purchased from Toyobo (Japan). A 1kb DNA ladder was from Dongsheng Co. Ltd. (Guangzhou, China). Enzymatic reactions were carried out under recommended conditions. All other chemicals used in the study were of molecular biology grade.
Asymmetric single-tube bridge PCR reaction system
Asymmetric single-tube bridge PCR was conducted in a 50μl mixture: 10 μM P1P2 1μl (200nM final), 0.1-1 μM P3P4 1 μl (2-20 nM final), 10μM P1R 1 μl (200nM final), DNA template appropriate quantity, 2 mM dNTP 5 μl , 25 mM MgSO4 2 μl, 10×KOD buffer 5 μl, KOD Plus 1 μl (1 unit), PCR-grade water 33μl. For Kan-Amp double-resistance reporter construction, the following DNA template was included: pIRES2-EGFP 50ng and pUC19 100ng. For porcine MSTN reporter vectors the following template was added: Hubei white pig genomic DNA 100ng, pGL3-basic/promoter plasmid 100ng. The PCR conditions were as follows: 95°C 2min, 30 cycles of (95°C for 15s, 55°C for 30s, 68°C for X min; X=total fused plasmid length/1kb). The PCR products were size fractionized by 1% agarose gel electrophoresis and documented by ChemiDoc™ XRS system (Bio-Rad, U.S.). As for the two-step PCR protocol, see details in (10).
PCR product processing, transformation and plasmid construction
The PCR products were digested by DpnI at 37°C for 1 or 2 hours to destroy methylated circular plasmids with the following reaction: PCR products, 26 μl, 10×Tango buffer 3 μl, DpnI 1 μl (1 unit). DpnI endonuclease works well in KOD Plus buffer so we did not typically take extra steps to purify PCR products. Aliquots of 5 μl digested PCR products were chemically transformed into 100 μl competent E.coli DH5α to generate recombinants. The Kan-Amp double resistance reporter plasmid was designed as: the 1.6kb kanamycin resistance gene cassette from pIRES2-EGFP was fused into the MCS region of pUC19, and an artificial ApaI restriction site was created to demarcate the insertion site. Porcine MSTN regulatory element reporters were created as follows: 2.3 kb and 3.8 kb porcine myostatin promoter sequences were fused into the upstream of Fluc gene of pGL3-basic reporter, while the 1.4 kb porcine myostatin terminator fragment was fused into the downstream of Fluc gene of pGL3-promoter plasmid, replacing the SV40 late poly(A) signal sequence. For the MSTN expression cassette plasmid, the 3.8 kb promoter, 1.4 kb terminator, and 0.72 kb EGFP cDNA were assembled in that order. PCR products were excised from 1% agarose gel with a scalpel and purified by a purification kit (Generay, Shanghai, China). Purified PCR products were eluted in 30 μl elution buffer (EB) and adjusted to 100 ng/μl. Their size and integrity were confirmed by electrophoresis of 2 μl products. LB plates were analyzed by Quantity One software (Bio-Rad, U.S.) to calculate the number of bacterial colonies. Randomly picked clones were sequenced using a ABI3730 DNA Analyzer (Applied Biosystems, Foster City, CA, U.S.) and the sequences were assembled and analyzed using DNAStar software (DNASTAR Inc., Madison, WI. U.S.).
Cell culture and transfection
A mouse myoblast C2C12 cell line was cultured in high glucose DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco) at 37°C and 5% CO2, saturated humidity. C2C12 differentiation was induced by DMEM with 2% horse serum (Invitrogen). 4×104 cells were seeded in a 24-well plate one day before transfection. For porcine MSTN regulatory element analysis, equal molar quantities of reporter constructs (Fluc served as a reporter) and internal control 250ng pRL-TK (Rluc served as a control) plasmids were co-transfected by Lipofectamine2000 (Invitrogen). Eight hours post transfection, the transfected wells were renewed with fresh medium for further culture. Cells were lysed 48h post transfection under proliferation conditions, or 5 days after differentiation. For MSTN expression cassette assay, equal molar quantities of pIRES2-EGFP, pUC19-5MSTN3 and pUC19-5EGFP3 were transfected into C2C12 cells. Forty-eight hours post transfection, fluorescence was captured using a Leica 4000B microsystem (Germany). All transfections were assessed in three independent experiments, each in triplicate.
A Dual Luciferase Reporter Assay System (Promega) was used to measure luciferase activity in a Glomax luminometer with slight deviations from the manufacturer’s instructions. In brief, 100 μl passive lysis buffer was used to disrupt cells on each well with gentle shaking at room temperature. 10 μl LARII solution was added to 20 μl cell lysate to initiate the primary scanning to measure Fluc luciferase activity. Shortly afterwards, 10 μl substrate solution was added into the same tube to initiate Rluc activity.
The Fluc luciferase activities were normalized to Rluc activities. Luciferase levels were reported as fold repression in activity over that observed in transfections with control treatments. The data represent the mean ± S.D. of three independent experiments and were analyzed by Student’s t-test. Differences below P < 0.05 were regarded as significant. EGFP fluorescence intensity was analyzed using ImageJ software.
The annotation of these reporters engineered in this work can be retrieved under accession number [Genbank: JN542719, JN542720, JN542721].