A Step-by-Step Procedure for Producing Germline Chimera in Chicken via Primordial Germ Cell Transplantation

Primordial germ cell (PGC)-mediated germline transmission is the most efficient method for transferring genetic information to the next generation in birds^1,2,3. Germline chimeras, defined as individuals carrying both donor- and host-derived germ cells, allow transmission of donor genetic material to offspring, supporting applications in transgenesis, functional genomics, and conservation of endangered species^4,5,6,7,8. Unlike approaches that rely on blastodermal cells, PGC-mediated strategies provide higher germline transmission efficiency and reproducibility, which makes them well-suited for precise genetic manipulation.

In avian species, PGCs originate in the central pellucida region of the blastoderm at Eyal-Giladi and Kochav (EGK) stage X⁹ and subsequently migrate to the germinal crescent by Hamburger and Hamilton (HH) stage 4¹⁰. Between HH stages 9 and 12, PGCs circulate via embryonic blood vessels before settling in the gonadal ridge^11,12,13. These distinct migratory pattern enables isolation of PGCs at multiple stages, including the germinal crescent, circulating blood, and gonads¹⁴. Notably, PGCs harvested from HH stage 26-28 gonads and transplanted into HH stage 14-17 recipient embryos have been shown to exhibit optimal germline competency and engraftment efficiency, providing a practical framework for standardized application of this protocol.

PGCs can be maintained and expanded in vitro while retaining germline identity and transmission capability, enabling their use for stable genetic modification. PGC-mediated germline chimera production has been used to generate transgenic birds expressing recombinant proteins, trace germline lineage, manipulate sex determination pathways, and confer resistance to infectious diseases^{6,15,16,17,18,19}. Furthermore, PGC transplantation facilitates interspecies germline transmission and conservation of endangered avian germplasm^{7,8,20,21,22,23}, and PGC cryopreservation enables long-term genetic resource preservation^24,25.

Although other stem cell types, including embryonic stem cells (ESCs)^26,27,28,29, induced pluripotent stem cells (iPSCs)^30,31,32, and spermatogonial stem cells (SSCs)^33,34, have been explored for avian germline modification, their germline transmission efficiency remains lower than that of PGC-based systems, which are currently regarded as the most reliable source of germline-competent cells.

In germline chimera studies, the choice of donor breed is critical for distinguishing donor-derived germ cells from host-derived cells. The Korean Ogye Chicken (KOC) possesses the recessive i/i allele responsible for black skin and feathers, which is easily distinguishable from the White Leghorn line carrying the dominant I/I allele^35,36. This visible difference allows for unambiguous identification of donor-derived offspring during germline transmission analysis, while also supporting biodiversity conservation due to the indigenous genetic value of KOC.

Overall, PGC-mediated germline chimera production provides a robust platform for avian biotechnology (Figure 1). By specifying the optimal donor (HH26-28) and recipient (HH14-17) stages and incorporating defined culture and validation steps, this protocol enables efficient germline transmission and provides a practical framework for the generation of transgenic and genome-edited birds, as well as the preservation of valuable avian genetic resources.

All procedures involving chickens were approved by the Institute of Laboratory Animal Resources, Seoul National University, South Korea (IACUC approval number: SNU-250226-4). Animals were maintained under controlled and hygienic conditions with sterilized water and feed provided. In addition, all chemical and biological waste generated during the procedures, including trypsin/EDTA solutions, PKH26-labeled cells, and embryonic or gonadal tissues, was handled and disposed of in accordance with the institutional biosafety and hazardous waste management guidelines of Seoul National University. All reagents and equipment used in this protocol are listed in the Table of Materials. PGCs media and reagents used for gonad sampling and cell culture from donor embryos must be sterile.

1. Isolation of chicken PGCs

1. Incubate fresh fertilized chicken eggs at the Eyal-Giladi and Kochav (EGK) stage X at 37 °C for 5.5 days until the embryos reach HH stages 26-28.
   NOTE: Maintain humidity at 60%-65% to ensure normal embryonic development.
2. Carefully isolate embryos from the eggs at HH stages 26-28 using sterile forceps.
3. Wash embryos with 1× PBS, then place the embryo on a charcoal plate and secure it with insect pins. Using a stereomicroscope, carefully dissect the paired embryonic gonads using sharpened forceps.
   NOTE: Handle the tissues gently to prevent damage and ensure accurate isolation of the gonads. Due to their close proximity to the mesonephric kidney at HH stages 26-28, a small amount of kidney tissue may be inadvertently collected; however, careful dissection under a stereomicroscope and selective culture conditions ensure that only PGCs proliferate, maintaining purity.
4. Transfer the paired gonads collected from three embryos into a 1.5 mL tube containing 500 µL of 0.05% trypsin/EDTA and incubate at 37 °C for 5 min to dissociate the cells.
5. Add 50 µL of FBS to inactivate the trypsin.
6. Centrifuge at 200 × g for 5 min at room temperature (approximately 20-25 °C). Carefully remove the supernatant without disturbing the cell pellet.
7. Resuspend the dissociated gonadal cells in 1 mL of 1× PBS.

2. In vitro proliferation of chicken PGCs

1. Prepare PGC culture medium (KO-DMEM supplemented with 20% FBS, 2% chicken serum, nucleosides, GlutaMAX, MEM non-essential amino acids, 2-mercaptoethanol, 10 mM sodium pyruvate, antibiotic-antimycotic, 10 ng/mL bFGF) and warm the medium to 37 °C.
2. Add dissociated gonadal cells collected from three embryos at HH stages 26-28 (approximately 100-150 PGCs per embryo^37,38) to 1 mL of pre-warmed culture medium per well and plate the cells onto a 12-well culture plate.
3. Gently rock the plate to ensure uniform distribution of the cells.
4. Maintain PGCs at 37 °C with 5% CO₂, monitor morphology and cell density daily. PGCs remain round, refractile, and typically floating, whereas somatic cells are adherent (Figure 2A).
   NOTE: The culture conditions selectively support the proliferation of PGCs, allowing PGCs to expand while somatic cells are gradually eliminated.
5. PGCs cultured in a 12-well plate are maintained by changing the culture medium every 3-4 days to support optimal proliferation and viability (Figure 2B).
6. Optionally, use PGC-specific markers (e.g., CVH, DAZL, NANOG, and POUV) to verify purity and conduct sex determination by PCR (Table 1).
   1. Prepare PGC samples for RNA extraction using the RNA Miniprep System according to the manufacturer's instructions.
   2. Perform RT-PCR (e.g., CVH, DAZL, NANOG, and POUV) or immunostaining using PGC-specific markers (SSEA1 and DAZL) according to standard protocols³⁹ (Figure 2C,D).
   3. Perform sexing PCR analysis according to standard protocols⁴⁰ (Figure 2E).

3. Validation of the germline competency of cultured PGCs using PKH26-labeling

1. Harvest PGCs when they reach the log-growth phase, typically at a density of approximately 1-5 × 10⁵ cells per well in a 12-well plate.
2. Pellet the cells by centrifuging at 200 × g for 5 min at room temperature (approximately 20-25 °C), resuspend in 1× PBS, and assess viability using trypan blue.
3. Prepare Diluent C from the PKH26 Red Fluorescent Cell Linker Kit for general cell membrane labeling and equilibrate it to room temperature (approximately 20-25 °C).
4. Reconstitute the PKH26 dye and prepare a working solution in Diluent C at a final concentration of approximately 1-2 µM.
5. Resuspend the PGC pellet in 1 mL Diluent C. Rapidly add 1 mL PKH26 solution (1:1 ratio), mix gently, and incubate for 2-5 min at room temperature (approximately 20-25 °C).
6. Rapidly add 1 mL PKH26 working solution to the cell suspension (1:1 ratio), gently mix by pipetting, and incubate at room temperature (approximately 20-25 °C) for 2-5 min.
   NOTE: Do not exceed the recommended incubation time.
7. Stop the staining by adding 2-4 times the volume of FBS or 1%-5% BSA in PBS.
8. Centrifuge at 200 × g, 5 min at room temperature (approximately 20-25 °C); wash three times with PGC culture medium or 1× PBS + 0.1% BSA.
9. Resuspend cells in injection buffer (PGC culture medium or 1× PBS + 0.1% BSA) at a concentration of 1-5 × 10⁴ to 1 × 10⁵ cells/µL. Keep them on ice briefly to preserve viability.
10. Verify the labeling under a fluorescence microscope before injection (Figure 3A).
11. Inject more than 3,000 viable labeled PGCs, counted using trypan blue, into HH stage 14-17 embryos and incubate until day 6 (Figure 3B).
12. Confirm colonization of recipient gonads by day 6 (Figure 3C).

4. Transplantation of donor PGCs into the recipient embryonic blood vessel

1. Incubate the recipient eggs to HH stage 14-17 (37 °C, 60-70% humidity, rocking at 45° every hour).
2. Prepare a glass capillary micropipette using borosilicate glass tubing (1.0 mm outer diameter, 0.5-0.75 mm inner diameter). Pull the capillary using a micropipette puller and polish the tip to an inner diameter of approximately 10-20 µm.
3. Create a small window (approximately 0.5 cm × 0.5 cm) at the pointed end of the egg to allow microinjection access while minimizing disturbance to the embryo.
   NOTE: Sterilize the eggshell surface with 70% ethanol prior to opening the window to prevent contamination.
4. Inject 2 µL of a suspension containing more than 3,000 PGCs in 1× PBS or HBSS into the dorsal aorta under a stereomicroscope.
   NOTE: Perform the injection under a stereomicroscope to avoid damaging the embryo or blood vessels, and carefully manipulate the microinjection needle to prevent injury.
5. Seal the egg window with paraffin film; position the egg pointed end down and incubate until hatching at 37.5 °C, 60-70% humidity, rocking at 45° every hour.
   NOTE: Maintain appropriate temperature and humidity to ensure embryo viability and successful development.

5. Validation of germline chimera

1. Collect the testis or ovary from the G0 chick post-hatching.
2. Extract genomic DNA from the collected gonadal tissues to confirm germline chimera formation. Perform genomic DNA extraction from the collected gonadal tissues as follows:
   1. Collect testis or ovary tissue, homogenize it in 300 µl of lysis buffer (2 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS) containing 1.5 µL of proteinase K, and incubate at 60 °C for h to ensure complete lysis.
   2. Add 100 µL of protein precipitation solution (7 M ammonium acetate), mix thoroughly, and centrifuge at 12,000 × g for 10 min at 4 °C.
   3. Transfer the supernatant (aqueous phase) to a new tube, add 500 µL of isopropanol to precipitate DNA, and centrifuge at 12,000 × g for 10 min at 4 °C.
   4. Discard the supernatant, add 1 mL of 70% ethanol, and centrifuge at 12,000 × g for 10 min at 4 °C.
   5. Remove the ethanol, air-dry the DNA pellet at room temperature for 10 min.
   6. Elute the DNA with 50 µL of RNase-free water and measure the DNA concentration and purity using a spectrophotometer (A₂₆₀/A₂₈₀ ≈ 1.8).
   7. Adjust DNA to 10-5 ng per PCR reaction.
3. Perform PCR amplification using species-specific primers that were designed (Table 1) under the following conditions: 94 °C for 3 min followed by 35 cycles of 94 °C for 30 s, 68 °C for 30 s, and 72 °C for 30 s, plus a final extension at 72 °C for 5 min (Figure 4A).
4. Collect semen from sexually mature G0 males, extract sperm DNA using the same protocol, and perform PCR using species-specific primers (Figure 4B).

In vitro proliferation and characterization of Chicken PGCs
PGC-containing whole gonadal cells were isolated from embryos at HH stages 26-28 and cultured under defined PGC culture conditions. Over time, gonadal stromal cells either adhered to the culture surface or underwent degradation, whereas PGCs remained in suspension and began to actively proliferate (Figure 2A). After approximately two weeks of culture, the PGC population expanded to more than 1 × 105 cells, demonstrating robust proliferation and selective enrichment of PGCs under these defined conditions (Figure 2B).

To confirm the germ cell identity of the cultured cells, RT-PCR analysis was conducted using germ cell-specific markers, and immunocytochemistry was performed. RT-PCR results demonstrated the expression of DAZL, CVH, NANOG, and POUV genes in the cultured PGCs (Figure 2C). Consistently, immunostaining revealed positive expression of SSEA-1 and DAZL proteins, confirming that the proliferating cells retained definitive PGC characteristics (Figure 2D). Furthermore, to verify the sex of proliferating PGCs cultured in vitro, PCR was performed using sex-specific markers (Table 1). The results confirmed that PGCs from all three independent batches were of male origin (Figure 2E).

Germline competency of cultured PGCs
To evaluate the germline competency of cultured PGCs, cultured PGCs were labeled with PKH26 fluorescent dye and verified under a fluorescence microscope to confirm successful membrane staining (Figure 3A). Trypan blue exclusion analysis demonstrated that PKH26 labeling did not affect cell viability, showing no significant difference compared with unlabeled PGCs (Figure 3B). The labeled donor PGCs were microinjected into the dorsal aorta of recipient embryos (both male and female) at HH stages 14-17, and the embryos were incubated until day 6 post-injection. For improved visualization during microinjection, the PGC suspension was mixed with Trypan blue (Figure 3C, left panel). Examination of recipient gonads at day 6 revealed the presence of PKH26-positive donor PGCs in both the left and right gonads, confirming that the transplanted PGCs successfully migrated and colonized the developing gonadal tissue (Figure 3C).

Generation of G0 germline chimeric chickens
When donor PGCs were derived from Korean Ogye Chicken (KOC, i/i) and transplanted into White Leghorn (WL, I/I) recipient embryos, the dominant WL phenotype allowed donor-derived offspring to be easily distinguished based on external appearance. The resulting G0 germline chimeras phenotypically exhibited the WL appearance, while their gonads contained germ cells originating from both KOC and WL. To assess the contribution of donor PGCs to the germline, genomic DNA (gDNA) was isolated from the gonads of hatched G0 chimeric chickens and analyzed by PCR using KOC- and WL-specific primers. PCR amplification confirmed the presence of donor-derived alleles in the recipient gonads (Figure 4A). Furthermore, semen collected from sexually mature G0 males was used to extract sperm gDNA, and the same PCR analysis was conducted. Donor-derived genetic sequences were detected in the sperm (Figure 4B), demonstrating that the transplanted PGCs successfully colonized the gonadal niche and contributed to functional germline formation.

Figure 1: Representative scheme of a primordial germ cells (PGCs)-mediated germline chimera production method. Gonads were collected from embryos at Hamburger and Hamilton (HH) stages 26-28, and whole gonads were dissected to isolate primordial germ cells (PGCs) under defined culture conditions, excluding gonadal stromal cells. Following validation with germ cell-specific markers, the cultured PGCs were transplanted into the dorsal aorta of recipient embryos at HH stages 14-17. This schematic illustrates the key steps of PGC isolation, in vitro expansion, and transplantation for germline chimera production. Please click here to view a larger version of this figure.

Figure 2: In vitro proliferation and characterization of chicken primordial germ cells (PGCs). (A) Representative images of primordial germ cells (PGCs) proliferation at day 2, day 6, and day 9 of in vitro culture. Scale bar = 100 µm. (B) Growth curve of PGCs cultured in vitro until day 14; P denotes passage number. (C) RT-PCR analysis showing expression of pluripotency markers (POUV, NANOG) and germ cell markers (DDX4, DAZL) in PGCs after three weeks of culture. (D) Immunocytochemical analysis showing positive expression of SSEA-1 and DAZL in PGCs cultured in vitro. Scale bar = 50 µm. E. Sex determination of cultured PGCs using sex-specific PCR. Please click here to view a larger version of this figure.

Figure 3: Distribution of PKH26-labeled donor primordial germ cells (PGCs) in the gonads of recipient chickens. (A) Donor primordial germ cells (PGCs) labeled with PKH26 (red) visualized under a fluorescence microscope. Scale bar = 100 µm. (B) Viability of PGCs before and after PKH26 labeling. Data are presented as mean ± SEM (n = 3); ns, not significant by t-test. (C) Donor PGCs labeled with PKH26 mixed with Trypan blue solution and transplanted into the dorsal aorta of recipient embryos at HH stages 14-17. Scale bar = 200 µm. Please click here to view a larger version of this figure.

Figure 4: Validation of donor primordial germ cells (PGCs)-derived progeny and germline chimerism. (A) PCR analysis using species-specific markers confirmed the presence of donor-derived PGCs in the gonads of G0 germline chimeras. The G0 germline chimera samples showed Korean Ogye Chicken (KOC)-specific amplicons, whereas only the recipient (WL) genotype was detected in the feather follicles. (B) Species-specific PCR analysis of sperm genomic DNA from G0 germline chimeric males. The G0 germline chimera in lane 3 was positive for both KOC-specific and WL-specific primers. Please click here to view a larger version of this figure.

Table 1: The primer sets used for reverse-transcription PCR.

The transplantation of cultured primordial germ cells (PGCs) into recipient embryos provides a reliable method for producing germline chimeras in chickens. This protocol capitalizes on the intrinsic germline potential of PGCs and improves experimental consistency compared to traditional blastoderm-based systems, which are prone to mosaicism and developmental variability. A critical advantage of this method is the selective in vitro expansion of PGCs under defined culture conditions, which maintains their germline identity. Throughout the culture period, PGCs retain characteristic morphology, exhibit high proliferation rates, and express germ cell-specific markers such as CVH and DAZL, confirming their undifferentiated state.

Precise control of the embryonic developmental stage is essential for successful engraftment. The HH stage 14-17 window is optimal because the dorsal aorta is fully accessible, and the gonadal ridges are receptive to colonization. If embryos are injected outside this window, donor cells may not reach the gonadal niche efficiently, resulting in decreased germline contribution. Therefore, staging should be verified using morphological landmarks rather than incubation time alone. Synchronizing incubation start time across eggs is recommended to minimize stage variation.

The injection technique is another critical factor. Misplacement of the injection needle or excessive pressure can lead to embryo mortality or donor cell loss. To avoid this, we recommend using beveled glass microcapillaries with calibrated inner diameters and restricting the injection volume to 1-2 µL. Real-time visualization of blood flow during injection can confirm correct intravascular delivery. Practicing mock injections using phenol red or dye is highly effective for operator training and improves reproducibility across laboratories.

Competition between endogenous and transplanted PGCs is a known limitation of this system. Donor cell engraftment may be reduced when endogenous PGCs occupy available niches. Increasing the number of transplanted cells (≥3 × 10³ cells per embryo) or using recipient lines with reduced endogenous PGC populations can significantly improve colonization efficiency^41,42,43,44. Previous studies have demonstrated that culturing PGCs for approximately 10 days results in germline transmission efficiencies exceeding 45%, indicating the high functional potential of in vitro-expanded PGCs³⁵. Consistent with these findings, this protocol confirmed germline contribution through the detection of donor-derived markers in sperm.

This PGC-based system is compatible with diverse genome editing tools, including CRISPR/Cas and transposon-based vectors, enabling the generation of transgenic and genome-edited avian models^{6,16,17,18,19}. These models have broad applications in developmental biology, disease resistance research, and bioreactors for protein production. Moreover, PGC transplantation strategies are highly applicable to avian conservation, facilitating interspecies germline transmission and restoration of endangered genetic resources^7,8,20.

In summary, this protocol provides a reliable and scalable method for avian germline modification. By emphasizing experimental timing, injection accuracy, and PGC quality control, researchers can achieve consistent germline transmission outcomes. The procedural refinements and troubleshooting strategies described here offer practical guidance for optimizing PGC transplantation and expanding its application across basic and applied avian biotechnology.