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

Summary

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

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

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

Protocol

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 Tabl....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x PBS	Hyclone	SH30256
2-Mercaptoethanol	Gibco	21985023
Antibiotic Antimycotic Solution (100X)	Gibco	15240062
Anti-DAZL antibody	Abcam	ab215718
Chicken Serum Sterile	Rockland	D102-00-0500
Deoxyribonuclease I from bovine pancreas	Sigma aldrich	9003-98-9
Drummond Microcaps Disposable Micropipets	Drummond Scientific Company	1-000-0500
EmbryoMax Nucleosides (100X)	Merck	ES-008-D
FBS (Fetal Bovine Seurm Characterized, US-Sourced, 500ml)	Hyclone	SH30919
GlutaMAX Supplement	Gibco	35050061
KnockOut DMEM	Gibco	10829018
MEM Non-Essential Amino Acids Solution	Gibco	11140050
Parafilm M Laboratory Film	Amcor	PM996
PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling	Sigma aldrich	PKH26GL-1KT
Purified anti-mouse/human CD15 (SSEA-1) Antibody	Biolegend	125602
Recombinant Human FGF basic/FGF2/bFGF (146 aa) Protein	R&D systems	233-FB
ReliaPrep RNA Miniprep Systems	Promega	Z6011
Sodium Pyruvate (100 mM)	Gibco	11360070
Trypan blue solution	Sigma	T8154-100ML
Trypsin-EDTA (0.5%), no phenol red	Gibco™	15400054