Fertile eggs for the derivation of wild-type (WT) PGCs were produced via artificial insemination using semen collected from WT White Leghorn (WL) male chickens and WT WL female chickens. Additionally, heterotypic transgenic males expressing the 3D8 single-chain variable fragment (scFv) gene [13] and WT WL female chickens were paired to generate fertile eggs carrying the 3D8 scFv transgene. To distinguish transgenic from WT embryos, embryonic blood was collected for polymerase chain reaction (PCR) screening. All embryos were continuously incubated after sampling, and only those identified as positive for the 3D8 scFv gene were used for the isolation and culture of transgenic PGCs. The primers used were: forward, CCT CTG CTA ACC ATG TTC ATG CCT TC; reverse, GCT AGT GAA TGT GTA TCC AGA AGC CTT. All poultry were housed and managed under standard conditions at the institutional poultry farm. The experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Institute of Animal Science (NIAS-2023-593), Republic of Korea, in accordance with ethical guidelines for animal research.

Freshly laid fertilized eggs were incubated at 37.5°C until the embryos reached 5.5 to 6.0 days of development. The embryos were washed twice with phosphate-buffered saline (PBS; Gibco) and placed on black wax-coated Petri dishes. The embryonic gonads were surgically removed using sterilized tweezers and transferred to 1.5 mL microcentrifuge tubes containing 400 μL of PBS supplemented with 0.5% bovine serum albumin (BSA; Sigma-Aldrich). Subsequently, 100 μL of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) was added (final concentration of 0.05%), and the samples were incubated at 37.5°C in 5% CO₂ for 10–15 minutes to dissociate the tissue. The digested suspension was diluted with 4.5 mL of PBS and centrifuged at approximately 120×g for 5 minutes, after which the pellet was resuspended in PGC culture medium. The cell suspension was seeded into a 6-well culture plate, and after 4–5 hours, nonadherent cells were transferred to new 6-well plates precoated with mitotic inactivated feeder cells.

The feeder cells used incubated SIM mouse embryo-derived thioguanine-resistant (STO) cells (CRL-1503), mouse embryonic fibroblast (3T3-L1) cells (CL-173), and Buffalo rat liver (BRL/3A) cells (CRL-1442), which were all purchased from the American Type Culture Collection (ATCC). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1× antibiotic-antimycotic (anti-anti; Gibco). Feeder cells were mitotically inactivated via treatment with 10 μg/mL mitomycin C (Roche) for 2 hours. The cells then washed thoroughly with PBS and detached using 0.05% trypsin-EDTA. Finally, inactivated feeder cells were frozen in Cell Banker Solution (AMSBIO) until use. For PGC culture, inactivated feeder cells were thawed and seeded 0.1% gelatin-coated 6-well culture plates.

The resuspended PGCs were cultured in knockout DMEM (KO-DMEM; Gibco) supplemented with 20% heat-inactivated FBS, 2% chicken serum (Gibco), 20% BRL/3A conditioned medium, 1× L-glutamine, 1× Minimum essential medium nonessential amino acids, 1× nucleotide mix, 1× antibiotic-antimycotic, and 1 mM sodium pyruvate. The BRL/3A cells for conditioned medium preparation were cultured in DMEM supplemented with 10% FBS and 1× anti-anti until approximately 90% confluence. The cells were then treated with mitomycin C, washed three times with PBS, and cryopreserved. For conditioned medium preparation, thawed cells were maintained in KO-DMEM for three days, and the collected supernatant was stored at −20°C, used at 20% (v/v) concentration. Growth factors, including 10 ng/mL basic fibroblast growth factor (bFGF), 10 ng/mL stem cell factor (SCF), 10 ng/mL insulin-like growth factor 1 (IGF-1), and 10 ng/mL activin A, were added to optimize the culture conditions. The cultures were maintained at 37.5°C in a 5% CO₂ atmosphere, with media replaced every 2–3 days. The cells were monitored regularly, and PGCs were transferred to fresh wells when they reached confluence. Transgenic 3D8 PGCs were also cultured using this system under the same conditions as WT PGCs, following identical media composition, feeder preparation, and maintenance procedures.

Primary isolated PGCs (denoted PGCs #1 and PGCs #2) were plated 4-well culture plates. The cells were fixed with 4% paraformaldehyde (Invitrogen) for 10 minutes and permeabilized using 0.5% Triton X-100 for 15 minutes at room temperature. After being blocked with 5% BSA, the cells were incubated overnight at 4°C with primary antibodies against stage-specific embryonic antigen-1 (SSEA-1; Thermo Scientific, 41-1200, 1:200) and deleted in azoospermia-like (DAZL; Thermo Scientific, BS-12245R, 1:200). Secondary antibodies were purchased from Abcam. Alexa Fluor 488-conjugated goat anti-mouse IgG (Abcam, ab150113, 1:500) and Alexa Fluor 647-conjugated goat anti-rabbit IgG (Abcam, ab150079, 1:500) were applied for 1 hour. Nuclei were counterstained with Hoechst 33258, and images were captured using a CoolLED’s pE-300-W microscope (Optinity, Korealabtech). To evaluate the early migration potential of cultured PGCs, the cells were labeled with the PKH67 Green Fluorescent Cell Linker Mini Kit (Merck) according to the manufacturer’s instructions. The labeling followed the standard PKH67 protocol, including dye incubation and quenching steps.

Total RNA was extracted from gonadal tissues and PGCs using an RNeasy Mini Kit (Qiagen). Complementary DNA (cDNA) was synthesized using the Superscript IV First-Strand Synthesis System (Invitrogen). Reverse transcription polymerase chain reaction (RT–PCR) was performed to assess the expression of PGCs-specific markers, including DAZL and DEAD-box helicase 4 (DDX4), and pluripotency-related genes such as octamer-binding transcription factor 4 (OCT4), SRY-box transcription factor 2 (SOX2), and Nanog homeobox (NANOG), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as the reference gene (Table 1). The primer sequences and PCR conditions were adopted from previously established protocols [14,15]. The amplified products were analyzed using agarose gel electrophoresis.

Recipient fertile eggs were incubated at 37.5°C until Hamburger and Hamilton stages 17–18 (2.5 days of development) [16]. For microinjection, eggs were briefly rotated by hand, and a 1.0–1.5 cm diameter hole was created at the pointed end of the shell. To assess the injection and incubation conditions, eggs were assigned to three experimental groups: (1) normal incubation without interventions, (2) incubation with an artificial hole at the pointed end, and (3) injection of DMEM into embryonic blood vessels through the artificial hole. PGCs were transferred with glass microcapillary needles (inner diameter: 45–55 μm) to inject 1–2 μL of KO-DMEM containing 1×10³ PGCs/μL into embryonic blood vessels. After injection, the hole was sealed with parafilm, and the eggs were incubated with the pointed end facing upward. Recipient embryos were maintained under standard incubation conditions until analysis.

To investigate the migratory capacity of PGCs, lentivirus particles expressing enhanced green fluorescent protein (EGFP; hereafter referred to as GFP) were produced using a lentiviral vector system (VectorBuilder). The viral stocks contained 1.0–1.1×10⁹ transduction units/mL. The primary isolated PGCs (1×10⁴ cells/well) were seeded in 24-well gelatin-coated culture plates and transduced with lentivirus supplemented with 10 μg/mL polybrene for 24 hours at various multiplicities of infection (MOIs: 0, 1, 10, 100). Following transduction, the cells were cultured in medium containing 0.6 μg/mL puromycin for 6 days and then maintained under puromycin-free conditions for 14 days to allow for recovery. To enhance selection stringency, the cells were cultured for an additional 30 days with 0.3 μg/mL puromycin. Successfully transduced cells, designated GFP-PGCs, were used for further analysis, including characterization and migration assessment.

To quantify GFP expression, GFP-PGCs were harvested and washed twice with PBS. The cells were fixed with 4% paraformaldehyde for 5 minutes at room temperature and permeabilized with 0.1% Triton X-100 for 2 minutes. After washing twice with PBS, 1×10⁴ cells per sample were analyzed using a FACSCalibur flow cytometer (BD Bioscience).

All the statistical analyses were conducted using GraphPad Prism 5.03 software (GraphPad). Differences in hatching rates among the experiment groups were evaluated using one-way ANOVA. A threshold of p<0.05 was considered to indicate statistical significance.

The cell survival and propagation of PGCs isolated through enzymatic dissociation and differential adhesion-based selection were assessed. Morphological comparison was performed after 30 days of culture on each feeder type. Among the three feeder cell types tested, STO feeder cells demonstrated the highest efficiency in supporting PGC expansion, yielding greater cell proliferation and stability than BRL/3A and 3T3-L1 feeder cells did (Figure 1A). The number of embryos used for isolation did not appear to impact cell yield, as PGCs derived from five embryos (PGCs #1) and two embryos (PGCs #2) exhibited comparable proliferation rates, reaching approximately 10⁵ cells within one month (Figure 1B). Immunofluorescence staining confirmed the expression of SSEA-1 and DAZL, which was consistent with the PGC-specific marker profiles (Figure 2A). RT–PCR analysis further confirmed the expression of DAZL, DDX4, OCT4, SOX2, and NANOG, indicating the maintenance of both germline-specific and pluripotency-associated transcriptional signatures (Figure 2B). To assess the migratory capacity of cultivated PGCs, the PGCs #2 group was visualized by PKH67 labeling and injected into recipient embryos. Seven days after the transfer, fluorescence microscopy confirmed the presence of labeled PGCs in the developing gonads, demonstrating successful homing to the germline niche (Figure 2C). These findings collectively indicate that PGCs obtained via this approach exhibit stable expansion while preserving their molecular and functional properties, which are essential for germline transmission.

To evaluate the proliferative ability of the PGCs following cryopreservation, PGCs #1 and PGCs #2 were frozen and thawed at passages 24 (p24) and 15 (p15), respectively. Both populations resumed proliferation after thawing, reaching cell counts exceeding 10⁵ (Figure 3A). The PGCs clusters remained intact, and continuous proliferation was observed from p25 to p30 for PGCs #1 and from p16 to p20 for PGCs #2 (Figure 3A). Quantitative analysis of cell numbers from p27 to p30 in PGCs #1 and p18 to p20 in PGCs #2 further confirmed their stable proliferation after cryopreservation (Figure 3B). These findings indicate that PGCs maintain their proliferative potential and viability after thawing, demonstrating their suitability for long-term preservation and subsequent applications.

To generate transgenic PGCs, PGCs #1 were transduced with a lentiviral vector carrying the GFP gene (Figure 4). GFP expression was first clearly detected at an MOI of 100 after the initial 6 days of puromycin selection (Figure 4A). The fluorescence signal persisted after 14 days of puromycin-free culture, and subsequent 30-day selection with 0.3 μg/mL puromycin resulted in a stable GFP-expressing population, designated GFP-PGCs (Figure 4B). Flow cytometry analysis confirmed that 59.5% of the GFP-PGCs expressed GFP (Figure 4D). Further characterization revealed that the germline competency of the GFP-PGCs was retained. Immunofluorescence and RT–PCR analysis confirmed the expression of PGC-specific markers and pluripotency-associated transcriptional profiles (Figures 5A, 5B). To assess in vitro functionality, GFP-PGCs were microinjected into recipient embryonic blood vessels, and three days posttransfer, GFP fluorescence was detected in primary cells isolated from recipient embryonic gonads, confirming successful migration and colonization within the germline niche (Figure 5C). These findings indicate that lentiviral-mediated transduction generated stable transgenic PGCs while preserving their germline characteristics and migratory potential.

To evaluate the feasibility of PGC transplantation, the hatching rate of embryos following microinjection with DMEM was assessed. Among the injected eggs (Group 3), 48.3% successfully hatched, confirming that microinjection does not critically impair embryo viability compared with that of eggs with only an artificial hole (Group 2), as no statistically significant difference was observed between the two groups (p>0.05) (Figure 6A). To verify whether our culture and transplantation protocol is applicable to transgenic PGCs, we used transgenic 3D8 PGCs, which were isolated and cultured from a 3D8 transgenic chicken. These PGCs express the 3D8 gene, and their transplantation into recipient embryos allowed us to validate the effectiveness of our method for isolating and culturing PGCs from transgenic chickens. At embryonic day 12.5 (E12.5), the gonads of developing embryos were collected for PCR analysis. The presence of the 3D8 scFv gene was successfully detected, confirming that the injected PGCs migrated to and colonized the recipient gonads (Figure 6B). These findings confirm that microinjection-based PGC transplantation supports embryo viability and enables stable germline integration of transgenic PGCs.