Experimental animals
All methods were performed in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC, SNU-210217–5) of Seoul National University.
Primary culture of zebra finch fibroblasts and chondrocyte-like cells
Primary ZEFs were prepared from 6-day-old embryos. Collected tissues were dissociated with 0.05% trypsin–EDTA and resuspended in Dulbecco’s Modified Eagle Medium (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (Hyclone) and 1 × antibiotic–antimycotic (Thermo Fisher Scientific, Waltham, MA, USA). Cells were seeded at a density of 1 × 106 cells per 100 mm dish. When grown to confluent layers, cells were considered to be at population doubling level (PDL) zero and were passaged to calculate the PDL. The PDL test was carried out as described previously [3]. Primary CLCs were cultured as described previously and were used as a positive control in RT-PCR analyses [32].
Retroviral infection and culture of immortalized cells
Retroviral vector particles were produced by the calcium phosphate co-precipitation method, as described previously [33]. Briefly, the retroviral pMXs-c-MYC vector (Addgene, Cambridge, MA, USA) containing the mouse c-MYC gene was introduced into GP293 cells along with the pVSV-G packaging plasmid (Invitrogen, Thermo Fisher Scientific Inc., Carlsbad, CA, USA). Target cells were exposed to retroviruses for 48 h. Transduced cells were maintained in the same growth medium as the primary cells. When grown to confluent layers, cells were considered to be at PDL zero and were passaged to calculate the cumulative PDL.
Senescence analysis
Primary ZEFs and immortalized cell lines were fixed and then stained using the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology, Danvers, MA, USA).
Cell cycle analysis
Cells were treated with 10 μg/mL RNase A (Invitrogen) for 30 min at 37 °C and 50 μg/mL propidium iodide (Millipore Sigma, Burlington, MA, USA) for 30 min at 4 °C. The cell cycle status was determined using the FACSCalibur system (BD Biosciences, San Jose, CA, USA) and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
Karyotype analysis
Primary ZEFs and immortalized cell lines were collected and karyotype analysis was performed as described previously [33].
RT-PCR and quantitative RT-PCR
Total RNA samples were isolated with Trizol (Invitrogen) and cDNA was synthesized using the Superscript III First-Strand Synthesis System (Invitrogen). RT-PCR was done using specific primers with conditions as follows: 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. Gene expression levels were measured using EvaGreen dye (Biotium, Hayward, CA, USA) and a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). All samples were normalized to internal controls and fold changes were calculated through relative quantification (2−△△Ct). Primer set information is listed in Additional file 2: Table S1.
Alcian blue staining
Primary ZEFs and immortalized cell lines were stained with 1% Alcian blue (Millipore Sigma) in 3% glacial acetic acid solution and were visualized under a fluorescence microscope.
Transfection and selection of primary and immortalized cells
Primary ZEFs and immortalized cell lines were transfected in serum-free medium with 1–2 μg of a piggyBac YFP-expressing vector and 1–2 μg of a transposase vector (CAGG PBase) using Lipofectamine 3000 reagent (Invitrogen). The transfection mixture was replaced with growth media 6 h after transfection. After culture for 2 more days, fluorescence levels were calculated using the FACSCalibur system and cell viability was measured by trypan blue staining (Millipore Sigma). To determine the most appropriate concentration for the selection of transfected cells, immortalized cells were treated with G418 at 0, 50, 100, 200, 300, 400, or 500 μg/mL, and the surviving cells were counted 1 week after treatment. Subsequently, transfected cells were treated with the selected concentration of G418 (300 μg/mL) for 1 week and the proportion of YFP-expressing cells was determined using the FACSCalibur system.
Gene targeting vector construction
Previously described all-in-one CRISPR/Cas9 plasmids [34] were used to target the zebra finch SOX9 gene. Guide RNA (gRNA) sequences targeting the gene were designed using Geneious Prime software, considering the on-target score. For insertion of gRNAs into the CRISPR/Cas9 plasmids (pX459), sense and antisense oligonucleotides were designed (Additional file 3: Table S2) and synthesized by Bionics (Seoul, South Korea). Annealing of sense and antisense oligonucleotides was carried out under the following thermocycling conditions: 95 °C for 30 s, 72 °C for 2 min, 37 °C for 2 min, and 25 °C for 2 min. The annealed oligonucleotides were ligated into the pX459 vector using the Golden Gate assembly method, and the constructed CRISPR/Cas9 vectors were validated by Sanger sequencing (Bionics).
Transfection and genomic DNA sequencing analysis of the immortalized cell line
To validate the mutation efficiencies of the gRNAs, CRISPR/Cas9 plasmids containing gRNAs (2 μg) and the piggyBac YFP-expressing vector were co-introduced into cells using Lipofectamine 3000 reagent (Invitrogen), and then G418 selection was performed for 1 week. Genomic DNA was extracted from transfected cells and regions encompassing the CRISPR/Cas9 target sites were amplified using specific primer sets (Additional file 3: Table S2). For sequencing analysis, the PCR amplicons were annealed into the pGEM-T Easy vector and analyzed by Sanger sequencing. The sequencing results were analyzed using Geneious Prime software.
TUNEL assay
Apoptotic cells were detected by a TUNEL assay using a TMR red in situ cell death detection kit (Roche, Basel, Switzerland), following the manufacturer’s instructions.
Statistical analysis
Comparisons of primary and immortal cells were done via Student’s t-tests using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA).