2.1 Antibody and reagent
Commercially available antibodies were used: anti-C-Kit (Southern Biotech), anti-DDX4/CVH (ab13840, Abcam); GAPDH Anti-Phosphoserine (SPM101, Abcam); Goat Anti-Mouse IgG (Cy3 labeled, Bio-Synthesis, Inc., Texas, USA;dilution ratio:1:100).
Recombinant Human Bone Morphogenetic Protein-4 (BMP4, cyt-361) is from PROSPEC Dulbecco's modified Eagle medium (DMEM. 41965062) and Fetal bovine serum (FBS. 10100-147) from Gibco; FuGENE® HD (E2311) and Dual-Luciferase® Reporter Assay System from Promega; Leukemia Inhibit Factor from mouse (mLIF L5158), Fibroblast Growth Factor-Basic human (bFGF, F0291), Stem Cell Human (SCF. S7901); 5-aza-2'-deoxycytidine and TSA from Sigma; Pierce RNA 3' end desthiobiotinylation kit from Thermo; RIP-Assay Kit (RN1001) from MBL; PARIS™ from Ambion; PAS / Glycogen Stain kit (D004-1-2) from Nanjing Jiancheng Bioengineering Institute.
2.2 Isolation and culture of ESCs, PGCs, and SSCs
The study employed freshly fertilized eggs from Rugao yellow chickens, provided by the Poultry Research Institute of the Chinese Academy of Agricultural Sciences. ESCs were derived from 0-d-old chicken embryos, PGCs from the genital ridge of 4.5-d-old embryos, and SSCs were from testes of 18.5-d-old embryos. Hatching conditions were 37.5°C and 65% relative humidity. For specific separation and cultivation, refer to Zhang's article(Zhang, Wang et al. 2017).
2.3 Isolation of nuclear and cytoplasmic RNA
Operations were performed according to the manufacturer's protocols of PARIS™ system Protein and RNA Isolation System; add 500 μL Cell Disruption Buffer to every 107 PGCs cells, and incubate on ice for 10 mins; After centrifugation at 500 ×g for 5 mins at 4℃, the supernatant is cytoplasm and the precipitate is the nucleus. After the sample is dissolved, RNA extraction is performed.
2.4 Preparation of LncPGCR constructs
Three shRNA knockdown target sites were designed and combined to the linear vector pGMLV-SC5. shRNA target sites sequence is shown in Supplementary Table.4. The constructed knockdown vector was transfected with DF1 for activity verification, and the most active vector was encapsulated with lentivirus; A pair of primers was designed to amplify the full-length CDS region of LncPGCR. The fragment was cloned into pcDNA3.0 plasmid digested with KpnI and EcoRI to construct an overexpression vector and verify its activity. PCR primers are shown in Supplementary Table.6.
2.5 Chicken embryo vascular injection
Under sterile conditions, use tweezers to open a round hole with a diameter of 1-1.5cm in the blunt end of the early embryo eggs (13-17HH) incubated for 48-58h. Find the position of the embryo under a stereomicroscope. Then, using a micropipette, fill the lentiviral vector with a final polybrene concentration of 8ng / µL or the pcDNA 3.0-PGCR vector wrapped with PEI into glass injection needles and inject them into the blood vessels of chicken embryos; then add 20µL of Streptomyces penicillin vaccinate to the injection site, and finally cross-sealed with medical tape to continue hatching; Chicken embryos were collected at 4.5d and observe the embryos under a stereo fluorescence microscope.
2.6 Quantitative Real-time PCR (qRT-PCR)
Total RNA was extracted using Trizol (TIANGEN, Beijing, China) and reverse-transcribed into cDNA with the Quantscript RT Kit (TIANGEN, Beijing, China). Gene expression was determined using an ABI PRISM 7500 fluorescent quantitative PCR instrument (Applied Biosystems, Carlsbad, California). qRT-PCR primers are shown in Supplementary Table 5, the internal reference gene: β-actin, the number of repetitions: n = 3.
2.7 Immunocytochemistry (ICC)
6d ESCs in each group were fixed with 4% paraformaldehyde for 30 minutes, and then treated with 0.5% TritonX-100 for 15 minutes. After blocking for 2 hours in the dark, and then added with Cvh antibody. After incubating at 37 ℃ for 2 hours, overnight at 4℃; Then adding anti-mouse IgG, incubate at 37 ℃ in the dark for 2 hours; Next, it was stained with 5ng / µL DAPI for 15min, and finally, slides were plated with glycerin(50% glycerol, 50% PBS), and the image was sealed. FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan) was used to observed the samples.
2.8 Flow cytometric
For flow cytometry analysis, the cultures were mildly trypsinized and harvested from 24 well plates. The cells were washed and resuspended in PBS buffer, then add Cvh antibody for labeling, incubate at 4℃ overnight or incubate at 37℃ for 1-2h; Finally, data were analyzed on a Flow cytometer (Becton Dickinson, San Jose, CA) with the Flowjo program.
2.9 PAS staining
The paraffin sections after dewaxing and rehydration were stained according to the PAS / Gycogen Stain kit (D004-1-2) of Nanjing Jiancheng Technology Co., Ltd.
2.10 Plasmids
The LncPGCR promoter region and each deleted fragment were designed and combined to the linear vector pGL3.0-basic. Primers are shown in Supplementary Table 1.
2.11 Dual-luciferase assay
pGL3/1033 and pLncPGCR-N1 plasmid was transfected into DF-1. Then 10μmol/L 5-aza-2’-deoxycytidine (5-Azadc) or 1μmol/L Trichostatin A (TSA) was added in the cells. After 24h, cells were collected and luciferin was added using Dual-Luciferase Reporter Assay (Promega, USA).
2.12 RNA pull-down
Use Ambion's in vitro transcription kit, Maxiscript T7, for in vitro transcription, and perform RNA pull-down experiments according to Thermo's kit instructions to adsorb proteins that interact with LncPGCR; The target proteins were separated on PAGE by vertical electrophoresis and then subjected to silver staining experiments. Design the sense and antisense PCR primers based on the LncPGCR sequence(Supplementary Table 6).
2.13 RNA immunoprecipitation (RIP) assays
RIP assays were performed using a GAPDH Anti-Phosphoserine (SPM101, Abcam), and operations were performed according to the manufacturer's protocols of RiboCluster ProfilerTM (RN1001, MBL)RIP-Assay kit. Western Blot was used to detect the expression of the phosphorylation level of GAPDH on the extracted cellular protein samples after RNA enrichment.
2.14 Data analysis
Relative gene expression was calculated using the 2−ΔΔCt method after PCR. All experiments were performed in triplicate, and the data are expressed as mean ± standard error. Significant differences between the groups were determined with two-sample t-tests in SPSS 18.0. (*, P<0.05, significant difference.**, P<0.01, extremely significant difference).GraphPad Prism7 software was used for mapping.