Method Article

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

DOI:

10.3791/69704

⸱

December 5th, 2025

 ,  , 
1Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, 2Department of International Agricultural Technology & Institute of Green Bioscience and Technology, Seoul National University

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Avian germline chimeras, generated by the transplantation of primordial germ cells (PGCs) into recipient embryos, enable applications in transgenesis, gene editing, and species conservation. This protocol details procedures for PGC isolation, in vitro expansion, microinjection, and donor verification, providing a reliable platform for producing germline chimeras and advancing avian biotechnology.

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The generation of germline chimeras in chickens via transplantation of primordial germ cells (PGCs) provides a robust and reproducible platform for avian transgenesis, genome editing, and species conservation. Traditional approaches using blastodermal cells from EGK stage X embryos are limited by low germline transmission efficiency due to early cell lineage segregation. In contrast, PGC-mediated strategies exploit the intrinsic germline competency of these unipotent cells, enabling reliable incorporation into recipient gonads. Here, we present a stepwise protocol for isolating PGCs from HH stage 26-28 embryos, maintaining and expanding them in vitro, and transplanting them into the dorsal aorta of HH stage 14-17 recipient embryos. Short-term engraftment is monitored using fluorescent markers such as PKH26, while long-term germline contribution is confirmed by PCR analysis of recipient gonads. This approach ensures consistent generation of germline chimeras while preserving the viability and functional competence of donor PGCs. Additionally, the method is compatible with genome editing tools such as CRISPR/Cas9 and transposon-based vectors, enabling the production of transgenic and gene-edited avian models. Beyond basic research, this strategy supports conservation efforts through interspecies germline transmission and genetic resource preservation. Overall, this protocol provides a comprehensive and reliable framework for manipulating the avian germline, offering a versatile platform for both fundamental and applied studies in poultry biotechnology.

Introduction

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Primordial germ cell (PGC)-mediated germline transmission is the most efficient method for transferring genetic information to the next generation in birds1,2,3. 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 species4,5,6,7,8. 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 ridge11,12,13. 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 diseases6,15,16,17,18,19. Furthermore, PGC transplantation facilitates interspecies germline transmission and conservation of endangered avian germplasm7,8,20,21,22,23, and PGC cryopreservation enables long-term genetic resource preservation24,25.

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 allele35,36. 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.

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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 embryo37,38) 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).

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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...

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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 germlin...

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have nothing to disclose.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) [RS-2024-00418297] and Cooperative Research Program for Agriculture Science and Technology Development [RS-2025-02213489] from the Korean Rural Development Administration.

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1x PBS HycloneSH30256
2-MercaptoethanolGibco21985023
Antibiotic Antimycotic Solution (100X)Gibco15240062
Anti-DAZL antibodyAbcamab215718
Chicken Serum SterileRocklandD102-00-0500
Deoxyribonuclease I from bovine pancreasSigma aldrich9003-98-9
Drummond Microcaps Disposable Micropipets Drummond Scientific Company1-000-0500
EmbryoMax Nucleosides (100X)MerckES-008-D
FBS (Fetal Bovine Seurm Characterized, US-Sourced, 500ml)HycloneSH30919
GlutaMAX SupplementGibco35050061
KnockOut DMEMGibco10829018
MEM Non-Essential Amino Acids SolutionGibco11140050
Parafilm M Laboratory FilmAmcorPM996 
PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane LabelingSigma aldrichPKH26GL-1KT
Purified anti-mouse/human CD15 (SSEA-1) AntibodyBiolegend125602
Recombinant Human FGF basic/FGF2/bFGF (146 aa) Protein R&D systems 233-FB
ReliaPrep RNA Miniprep SystemsPromegaZ6011
Sodium Pyruvate (100 mM)Gibco11360070
Trypan blue solutionSigmaT8154-100ML
Trypsin-EDTA (0.5%), no phenol redGibco™15400054

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. van de Lavoir, M. C., et al. Germline transmission of genetically modified primordial germ cells. Nature. 441 (7094), 766-769 (2006).
  2. Macdonald, J., Glover, J. D., Taylor, L., Sang, H. M., McGrew, M. J. Characterisation and germline transmission of cultured avian primordial germ cells. PLoS One. 5 (11), e15518(2010).
  3. Choi, J. W., et al. Basic fibroblast growth factor activates MEK/ERK cell signaling pathway and stimulates the proliferation of chicken primordial germ cells. PLoS One. 5 (9), e12968(2010).
  4. Macdonald, J., et al. Efficient genetic modification and germline transmission of primordial germ cells using piggyBac and Tol2 transposons. Proc Natl Acad Sci U S A. 109 (23), E1466-E1472 (2012).
  5. Park, T. S., Han, J. Y. PiggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens. Proc Natl Acad Sci U S A. 109 (24), 9337-9341 (2012).
  6. Lee, H. J., et al. DMRT1 gene disruption alone induces incomplete gonad feminization in chicken. FASEB J. 35 (9), e21876(2021).
  7. Kang, S. J., et al. Reproduction of wild birds via interspecies germ cell transplantation. Biol Reprod. 79 (5), 931-937 (2008).
  8. Wernery, U., et al. Primordial germ cell-mediated chimera technology produces viable pure-line Houbara bustard offspring: potential for repopulating an endangered species. PLoS One. 5 (12), e15824(2010).
  9. Eyal-Giladi, H., Kochav, S. From cleavage to primitive streak formation: A complementary normal table and a new look at the first stages of the development of the chick. I. General morphology. Dev Biol. 49 (2), 321-337 (1976).
  10. Hamburger, V., Hamilton, H. L. A series of normal stages in the development of the chick embryo. J Morphol. 88 (1), 49-92 (1951).
  11. Ukeshima, A., Yoshinaga, K., Fujimoto, T. Scanning and transmission electron microscopic observations of chick primordial germ cells with special reference to the extravasation in their migration course. J Electron Microsc (Tokyo). 40 (2), 124-128 (1991).
  12. Fujimoto, T., Ukeshima, A., Kiyofuji, R. The origin, migration and morphology of the primordial germ cells in the chick embryo. Anat Rec. 185 (2), 139-145 (1976).
  13. Meyer, D. B. The migration of primordial germ cells in the chick embryo. Dev Biol. 10, 154-190 (1964).
  14. Kim, Y. M., Han, J. Y. The early development of germ cells in chicken. Int J Dev Biol. 62 (1-2-3), 145-152 (2018).
  15. Park, T. S., et al. Deposition of bioactive human epidermal growth factor in the egg white of transgenic hens using an oviduct-specific minisynthetic promoter. FASEB J. 29 (6), 2386-2396 (2015).
  16. Kim, Y. M., et al. The transgenic chicken derived anti-CD20 monoclonal antibodies exhibits greater anti-cancer therapeutic potential with enhanced Fc effector functions. Biomaterials. 167, 58-68 (2018).
  17. Rengaraj, D., et al. Dissecting chicken germ cell dynamics by combining a germ cell tracing transgenic chicken model with single-cell RNA sequencing. Comput Struct Biotechnol J. 20, 1654-1669 (2022).
  18. Park, J. S., et al. Production of recombinant human IgG1 Fc with beneficial N-glycosylation pattern for anti-inflammatory activity using genome-edited chickens. Commun Biol. 6 (1), 589(2023).
  19. Idoko-Akoh, A., et al. Creating resistance to avian influenza infection through genome editing of the ANP32 gene family. Nat Commun. 14 (1), 6136(2023).
  20. Ono, T., Yokoi, R., Aoyama, H. Transfer of male or female primordial germ cells of quail into chick embryonic gonads. Exp Anim. 45 (4), 347-352 (1996).
  21. Reynaud, G. The transfer of turkey primordial germ cells to chick embryos by intravascular injection. J Embryol Exp Morphol. 21 (3), 485-507 (1969).
  22. Liu, C., et al. Production of chicken progeny (Gallus gallus domesticus) from interspecies germline chimeric duck (Anas domesticus) by primordial germ cell transfer. Biol Reprod. 86 (4), 101(2012).
  23. van de Lavoir, M. C., et al. Interspecific germline transmission of cultured primordial germ cells. PLoS One. 7 (5), e35664(2012).
  24. Naito, M., Tajima, A., Tagami, T., Yasuda, Y., Kuwana, T. Preservation of chick primordial germ cells in liquid nitrogen and subsequent production of viable offspring. J Reprod Fertil. 102 (2), 321-325 (1994).
  25. Tajima, A., Naito, M., Yasuda, Y., Kuwana, T. Production of germline chimeras by transfer of cryopreserved gonadal primordial germ cells (gPGCs) in chicken. J Exp Zool. 280 (3), 265-267 (1998).
  26. Carsience, R. S., Clark, M. E., Verrinder Gibbins, A. M., Etches, R. J. Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos. Development. 117 (2), 669-675 (1993).
  27. Petitte, J. N., Liu, G., Yang, Z. Avian pluripotent stem cells. Mech Dev. 121 (9), 1159-1168 (2004).
  28. Petitte, J. N., Clark, M. E., Liu, G., Verrinder Gibbins, A. M., Etches, R. J. Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells. Development. 108 (1), 185-189 (1990).
  29. Pain, B., et al. Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities. Development. 122 (8), 2339-2348 (1996).
  30. Rossello, R. A., et al. Mammalian genes induce partially reprogrammed pluripotent stem cells in non-mammalian vertebrate and invertebrate species. Elife. 2, e00036(2013).
  31. Lu, Y., et al. Avian-induced pluripotent stem cells derived using human reprogramming factors. Stem Cells Dev. 21 (3), 394-403 (2012).
  32. Kim, Y. M., Park, Y. H., Lim, J. M., Jung, H., Han, J. Y. Technical note: Induction of pluripotent stem cell-like cells from chicken feather follicle cells. J Anim Sci. 95 (8), 3479-3486 (2017).
  33. Han, J. Y., et al. Production of quail (Coturnix japonica) germline chimeras by transfer of Ficoll-enriched spermatogonial stem cells. Theriogenology. 154, 223-231 (2020).
  34. Pramod, R. K., et al. Isolation, characterization, and in vitro culturing of spermatogonial stem cells in Japanese Quail (Coturnix japonica). Stem Cells Dev. 26 (1), 60-70 (2017).
  35. Park, T. S., et al. Improved germline transmission in chicken chimeras produced by transplantation of gonadal primordial germ cells into recipient embryos. Biol Reprod. 68 (5), 1657-1662 (2003).
  36. Chang, I. K., et al. Production of germline chimeric chickens by transfer of cultured primordial germ cells. Cell Biol Int. 21 (8), 495-499 (1997).
  37. Nakamura, Y., et al. Migration and proliferation of primordial germ cells in the early chicken embryo. Poult Sci. 86 (10), 2182-2193 (2007).
  38. Kuwana, T. Migration of Avian Primordial Germ Cells toward the Gonadal Anlage: (avian/PGC/migration/presumptive gonad/chimera). Dev Growth Differ. 35 (3), 237-243 (1993).
  39. Jung, K. M., Kim, Y. M., Ono, T., Han, J. Y. Size-dependent isolation of primordial germ cells from avian species. Mol Reprod Dev. 84 (6), 508-516 (2017).
  40. Ogawa, A., et al. Molecular characterization and cytological mapping of a non-repetitive DNA sequence region from the W chromosome of chicken and its use as a universal probe for sexing carinatae birds. Chromosome Res. 5 (2), 93-101 (1997).
  41. Park, K. J., et al. Gamma-irradiation depletes endogenous germ cells and increases donor cell distribution in chimeric chickens. In Vitro Cell Dev Biol Anim. 46 (10), 828-833 (2010).
  42. Nakamura, Y., et al. Germline replacement by transfer of primordial germ cells into partially sterilized embryos in the chicken. Biol Reprod. 83 (1), 130-137 (2010).
  43. Song, Y., D'Costa, S., Pardue, S. L., Petitte, J. N. Production of germline chimeric chickens following the administration of a busulfan emulsion. Mol Reprod Dev. 70 (4), 438-444 (2005).
  44. Lee, H., et al. Compensatory proliferation of endogenous chicken primordial germ cells after elimination by busulfan treatment. Stem Cell Res Ther. 4 (6), 136(2013).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Primordial Germ CellsPGC TransplantationAvian TransgenesisGenome EditingChicken EmbryosInterspecies Germline TransmissionCRISPR Cas9Fluorescent MarkersGonad PCR Analysis

Related Articles