Screening and Identification of Ghrdm Positive Cell Lines

Screening and Identification of Ghrdm Positive Cell Lines

Re-submission to Transgenic Research (TRAG-D-15-00048)


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 6L : 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 95C; 35 cycles of 95C at 30 sec., 60C at 30 sec., and 72C at 1min; final extension at 72C 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. 95C; 40 cycles 95C at 15 sec., 60C 34 sec., and 72C 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 100C 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 4C 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).