The care and experimental use of zebra finches were approved by the Institute of Laboratory Animal Resources, Seoul National University, Korea. All procedures, including zebra finch maintenance, reproduction, and sample collection, were governed by standard operating protocols according to a standard management program at the University Animal Farm, and the Animal Genetic Engineering Laboratory, Seoul National University.
All experimental procedures and care of zebra finches were approved by the Institute of Laboratory Animal Resources, Seoul National University. All methods were performed in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and approved by the Institutional Animal Care and Use Committee (IACUC, SNU-200305-2-1) of Seoul National University.
Culture of zebra finch PGCs
Gonads retrieved from zebra finch embryos at HH 28 were enzymatically digested and then cultured in vitro using a previously reported method21. Briefly, gonadal cells were suspended in PGC culture medium, which comprised knockout Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 2% chicken serum (Millipore Sigma, Burlington, MA, USA), 1 × nucleosides (Millipore Sigma), 2 mM l-glutamine (Thermo Fisher Scientific), 1 × nonessential amino acids, β-mercaptoethanol, 10 mM sodium pyruvate, 1 × antibiotic–antimycotic reagents (Thermo Fisher Scientific), and 10 ng/mL human basic fibroblast growth factor (Millipore Sigma). The PGCs-enriched suspension was sub-cultured every 4–5 days for about 1 month, and a fraction of adherent cells was used as a feeder layer.
Sexing of embryos was performed by PCR amplification of the chromodomain-helicase-DNA-binding protein (CHD)1 gene using genomic DNA extracted from extraembryonic tissues and gene-specific primers (Supplementary Table 1)41.
Density gradient centrifugation
Density gradient centrifugation of testicular cells was performed using a previously reported method with some modifications20. Testes were surgically dissected from sexually mature zebra finches (6 months old) and dissociated with 0.05% trypsin–EDTA. The cell suspension was filtered through a 40 µm nylon cell strainer. Then, 4 mL of the cell suspension in phosphate-buffered saline (PBS) was mixed with 3 mL of Ficoll-Paque PLUS solution (GE Healthcare Life Science, Chicago, IL, USA) and centrifuged at 800 × g for 30 min. After centrifugation, cells from differently layered fractions (top and bottom layers) were harvested and washed three times with PBS containing antibiotic–antimycotic reagents. Cells were pelleted and resuspended in culture medium, which comprised DMEM supplemented with 10% fetal bovine serum, 1 × nonessential amino acids, 15 ng/mL glial cell-derived neurotrophic factor (PeproTech, Rocky Hill, NJ, USA), 10 ng/mL basic fibroblast growth factor, 0.55 mM β-mercaptoethanol, 2 mM l-glutamine, 1 µM sodium pyruvate, and 1 × antibiotic–antimycotic solution at 37 °C in a humidified CO2 incubator containing 5% CO2. Cells were used for characterization or transfection at 1 day after seeding, and the transfected cells were maintained up to 4 days in vitro.
Immunocytochemical analysis of zebra finch PGCs and SSCs
Zebra finch PGCs and SSCs were dried on glass slides, fixed in 4% paraformaldehyde for 10 min, washed three times with PBS, and permeabilized with 0.1% Triton X-100 for 10 min. After three washes with PBS, cells were blocked with blocking buffer (PBS containing 5% goat serum and 1% bovine serum albumin) for 1 h and then incubated with an anti-DAZL antibody42 at 4 °C overnight. After three washes with PBS, cells were incubated with an Alexa Fluor 488-conjugated secondary antibody for 1 h at room temperature. Cells were finally mounted using ProLong Gold antifade reagent with DAPI and analyzed under a confocal fluorescence microscope (Carl Zeiss GmbH, Jena, Germany).
RT-PCR and quantitative RT-PCR
Total RNA samples were prepared using TRIzol reagent (Thermo Fisher Scientific) and reverse-transcribed into cDNAs using a SuperScript III Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. cDNAs were amplified by PCR using primer sets for the predicted zebra finch DEAD-box helicase 4 (DDX4), deleted in azoospermia-like (DAZL), POU domain class 5 transcription factor 3 (POUV), Nanog homeobox (NANOG), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes. PCR reactions comprised 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). Relative gene expression was quantified using the following formula: 2−ΔΔCt, where ΔΔCt = (Ct of the target gene − Ct of GAPDH)top layer − (Ct of the target gene − Ct of GAPDH)bottom layer. The primer sets used for RT-PCR and quantitative RT-PCR are listed in Supplementary Table 2.
Scanning electron microscopy
PGCs were fixed in 2% glutaraldehyde and postfixed in 1% osmium tetroxide at 4 °C for 2 h. After dehydration in a graded series of increasing concentrations of ethanol, the samples were immersed in hexamethyldisilazane and then dried. The samples were coated with gold palladium using a Leica EM ACE200 low-vacuum coater (Leica Microsystems, Buffalo Grove, IL, USA) and observed using a Sigma field emission scanning electron microscope (Carl Zeiss GmbH).
Migration assay of zebra finch PGCs
About 500 PGCs were injected into the dorsal aorta of zebra finch embryo at HH 13–16. The eggs were sealed with medical-grade silicone adhesive (Kwik-Cast, World Precision Instruments, Sarasota, FL, USA) and further incubated until HH 28. Fluorescent cells in recipient embryonic gonads were detected.
Transfection of PGCs and SSCs
A total of 5 × 105 PGCs cultured for 15–20 days or 1 × 106 SSCs cultured for 1 day after isolation were transfected with 1–2 μg of the GFP expression vector in a 12-well culture plate using an Amaxa Nucleofector (V buffer and X-001 program) or Lipofectamine 2000 reagent (Invitrogen). The nucleofection program was selected by testing three nucleofection programs (A-023, L-029, and X-001) using SSCs. PGCs and SSCs were also transduced with lentivirus (1 × 109 VP/mL) or adenovirus (1 × 1010 VP/mL) purchased from Vigene Bioscience (Vigene Bioscience, Rockville, MD, USA) at a multiplicity of infection (MOI) of 100. Cell viability was calculated as the number of viable cells divided by the total number of cells within the grid on the hemacytometer using trypan blue staining (Millipore Sigma). Transfected or transduced cells were resuspended in PBS and analyzed with a FACSCalibur system (BD Biosciences, San Jose, CA, USA). Subsequent analyses were performed using FlowJo software (Treestar, Ashland, OR, USA).
Primary culture of zebra finch embryonic fibroblasts (ZEFs)
The primary culture of ZEFs was prepared from the muscles of 6-day-old zebra finch embryos43. Several embryos (n = 3–5) were extracted from fertilized eggs, their head, limbs, and organs were removed, and the remaining contents were minced. Single-cell populations were obtained by 0.05% trypsin–EDTA treatment and maintained in DMEM (Hyclone) containing 10% fetal bovine serum (Hyclone) and 1 × antibiotic–antimycotic reagents (Thermo Fisher Scientific). Cells were seeded at a density of 5 × 105 cells/well in a 12-well culture plate and grown at 37℃ in an incubator containing 5% CO2.
The CRISPR/Cas9 vector targeting the ADNP gene was constructed using the pX459 vector, as previously reported44. The gRNA sequences targeting ADNP gene was designed by Geneious prime software considering on-target score (Supplementary Fig. 4C). To insert the gRNA sequence into the CRISPR/Cas9 vector, sense and antisense oligonucleotides were synthesized (Bionics, Seoul, South Korea) (Supplementary Table 3) and annealed using the following thermocycling conditions: 30 s at 95 °C, 2 min at 72 °C, 2 min at 37 °C, and 2 min at 25 °C. 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. Adenoviruses carrying hCas9 and ADNP gRNA sequences were produced and purified by ViGene Biosciences according to their standard protocol (CsCl gradient purification and plaque assay) and the vector designated as pAV-U6-gRNA-CBh-hCas9-EGFP.
Transfection and genomic DNA sequencing of ZEFs
To validate the mutation efficiency of the designed gRNA sequence targeting the ADNP gene, dissociated ZEFs (1 × 106) were resuspended in nucleofection solution containing 10 μg of CRISPR/Cas9 plasmids and nucleofection was performed using the X-001 program. Genomic DNA was extracted from transfected cells. Genomic regions encompassing the CRISPR/Cas9 target sites were amplified using specific primer sets (Supplementary Table 3). The PCR amplicons were annealed into the pGEM-T Easy Vector and sequenced by Sanger sequencing (Bionics). The sequencing results were analyzed using Geneious Prime software.
Genome-editing of zebra finch PGCs in vitro
A total of 5 × 105 PGCs cultured for 15–20 days were transduced with the adenovirus produced by Vigene Bioscience at a MOI of 100. Adenovirus for genome-editing was produced by transfecting 293A cells with a designed plasmid (pAV-U6-gRNA-CBh-hCas9-EGFP) by lipofection, and then purified by iodixanol gradient ultracentrifugation and tittered by IHC-immunohistochemistry (Vigene Bioscience). Genomic DNA was extracted from the cells 4 days after transduction. Genomic regions encompassing the CRISPR/Cas9 target sites were amplified using specific primer sets (Supplementary Table 3). The PCR amplicons were annealed to the pGEM-T Easy Vector and sequenced by Sanger sequencing (Bionics), and the sequencing results were analyzed using Geneious Prime software.
Analysis of off-target mutations in edited PGCs
Potential off-target sites were screened on the basis of high homologous scores, using the CCTop online prediction tool, and the 3 closest off-target sites were selected. These off-target sites shared 15–16 matched nucleotides with up to 11 consecutive matched sequences of the 20-base pair gRNA sequences (Supplementary Fig. 6A). The PCR amplification of the potential off-target regions using genomic DNAs from edited PGCs was conducted. The PCR amplicons were annealed to the pGEM-T Easy Vector and sequenced by Sanger sequencing (Bionics), and the sequencing results were analyzed using Geneious Prime software.
Transplantation of edited PGCs in vivo
About 1000 transduced PGCs were injected into the dorsal aorta of zebra finch embryo at HH 13–16. After injection, the eggs were sealed with medical-grade silicone adhesive (Kwik-Cast, World Precision Instruments) and further incubated until HH 28. Fluorescent cells in the recipient embryonic gonads were quantified under a fluorescence microscope.
Immunohistochemical analysis of embryonic gonads
Embryonic gonads with mesonephric tissues of zebra finch embryos at HH 28 were paraffin-embedded and sectioned (thickness, 8–10 μm). After deparaffinization, sections were washed with PBS and blocked with blocking buffer (PBS containing 5% goat serum and 1% bovine serum albumin) for 1 h at room temperature. Sections were then incubated with a rabbit anti-GFP primary antibody (Thermo Fisher Scientific) at 4 °C overnight. After washing with PBS, sections were incubated with an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific) for 1 h at room temperature. After washing, sections were mounted using ProLong Gold antifade reagent with DAPI and visualized using a confocal fluorescence microscope (Carl Zeiss GmbH).
Relative expression of genes was compared between the top and bottom fractions separated by Ficoll density gradient centrifugation using the Student’s t-test with GraphPad Prism statistical software (GraphPad Software, La Jolla, CA, USA). The transfection efficiency and cell viability using different transfection methods were compared by an ANOVA and Bonferroni’s Multiple Comparison Test using GraphPad Prism statistical software. All values are means ± standard deviation (n = 3).
Approval of animal experiments
All experimental procedures and care of zebra finches were approved by the Institute of Laboratory Animal Resources, Seoul National University. All methods were performed in accordance with the guidelines and regulations of the Institutional Animal Care and Use Committee of Seoul National University (IACUC, SNU-200305-2-1). All procedures, including zebra finch maintenance, reproduction, and sample collection, were governed by standard operating protocols according to a standard management program at the University Animal Farm, Seoul National University and the Animal Genetic Engineering Laboratory, Seoul National University.