Re-submission to Transgenic Research (TRAG-D-15-00048)
SUPPLEMENTARY MATERIALS AND METHODS
Screening and identification of GHRdm positive cell lines
Porcine fetal fibroblast (PFFs) were established from day 35 WZS embryos. Fibroblasts were cultured in DMEM media supplemented with 15% FBS, 1% glutamine, 1% NEAA and bFGF (5ng/ml). WZS PFFs cells were seeded into a six-well cell culture dish (BD, USA, cat. 353046) at 2×105 cells/well and transfected at 60% confluency with the FuGENE HD transfection reagent at ratio 6L : 2μg DNA (Roche,Switzerland, cat. 04709691001). Subsequently, the cells were re-seeded into six 10 cm dishes at 90% confluency and positively selected with G418 (500μg/ml) for two weeks. G418-resistant and eGFP-positive cell colonies were isolated and passaged into two 24-well plates at 80～90% confluency to be used for PCR and RT-PCR screening and as nuclear donor cells for handmade cloning (HMC).
PCR and RT-PCR screening
Genomic DNA was extracted from G418-resistant / eGFP-positive cell clones or tissue samples by use of the E.Z.N.A.Tissue DNA Kit (OMEGA, USA, cat. D3396-01). PCR was performed on 50 ng extracted DNA using 20 pmol GHR-F/R primer and 0.5 unit Ex Taq (Takara, Japan, cat. RR001A). The PCR program was as follows: 5 min. at 95C; 35 cycles of 95C at 30 sec., 60C at 30 sec., and 72C at 1min; final extension at 72C for 10min. From the PCR product 5μL was electrophoresed on a 1.2% agarose gel.
Total RNA was extracted from G418-resistant / eGFP-positive cell clonesor tissue samples by use of the Trizol reagent (Invitrogen, USA, cat. 15596-018). Subsequently, cDNA was synthesized with PrimeScriptTM 1stStrand cDNA Synthesis Kit (TaKara, Japan, cat. D6110A) employing 1μg of total RNA. The cDNA product was used for semi-quantitative or quantitative real-time PCR analysis using SYBR Premix Ex Taq(TaKara, Japan, cat. DRR420A). All reactions were performed in a total volume of 20 μL (10 μL 2×SYBR Premix Ex Taq, 0.4 μL10 μM primer, 0.4 μL ROX reference dye, 2 μL cDNA, and 6.8 μL nuclease-free water) and run the Applied Biosystems 7500 instrument. Real-time PCR conditions were as follows: 30 sec. 95C; 40 cycles 95C at 15 sec., 60C 34 sec., and 72C 40 sec. Primers were GHRdm-F/R, QWGHR-F/R and pGAPDH-F/R.
Genotyping of piglets
Genomic DNA and total RNA was extracted from tail clips taken of each newborn piglet at the age of 2 weeks. Transgene identification was carried out by PCR and RT-PCR as described above.
Western blot analysis
About 30mg tail tissue isolated from the newborn piglets were lysed in RIPA Lysis buffer (Beyotime, China, cat. P0013C)containing 1% PMSF (Phenylmethanesulfonyl fluoride). Protein lysates mixed with the 5×SDS PAGE buffer were incubated at 100C for 5 min, separated on 10% SDS PAGE gels and finally transferred to PVDF membranes by means of the Mini-ProteanTetra System (BIO-RAD, USA, cat. 165-8001). The membranes were incubated at 4C with a 1:1000 diluted anti-human GHR murine mAb (Abcam,UK, cat. MM0320-6J33) or a 1:3000 diluted anti-rat -actin murine mAb (Beyotime, China, cat.AA128). Afterwards, the membranes were incubated with HRP-conjugated goat anti-mouse secondary Ab (Beyotime, China, cat.A0216) for 2 hours at RT and visualized using the NovexECL HRP chemiluminescent Substrate Reagent kit (Invitrogen, USA, cat. WP20005).
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