Animal experiments

This study was performed in two centers; INSERM, INRAE, Stem Cell and Brain Research Institute, Claude Bernard University Lyon 1, Lyon, France and Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran. Fertilized eggs and recipient embryos of Hubbard and Bovans were obtained from Elevage Grand Buisson, France and from Seamorgh Company, Iran, respectively. cPGCs were collected from 2.5-day-old embryos, Hamburger–Hamilton (HH) stages 14–16, of both strains. Using chicken embryos before one-third of the incubation period did not require ethical approval. The study was conducted following the ARRIVE guidelines.

Culture media for cPGCs

To cultivate and maintain cPGCs, we utilized two media formulations: CSM and OTM. The base cPGC medium was prepared as described in our previous study^7,11. CSM was created by supplementing the base medium with 0.5% (v/v) chicken serum. For OTM, we substituted chicken serum with 10 µg/mL ovotransferrin (Sigma). All media components were sourced from Thermo Fisher Scientific, USA unless otherwise specified (Table 1).

Isolation and long-term culture of PGCs

Primary cultures of cPGCs were established from both Bovans and Hubbard embryos. The details of the isolation and cultivation methods of cPGCs were as described in our previous study^7,11. Briefly, Embryonic blood (1 µL) was isolated from the dorsal aorta of stage HH 14–16 embryos and cultured in 100 µL of CSM or OTM media in 96-well plates, as previously explained^7,11. The sex of embryos was determined from tissue samples. After 14 days, proliferated cPGCs were transferred to 48-well plates for expansion and long-term culture. The culture medium was refreshed every two days throughout the experiment. Established cell lines were defined as those exceeding 100 days in culture (100-day-old cPGCs). Male cPGC lines from both strains, cultured in either CSM or OTM for 100 days, were used for subsequent experiments.

Proliferation assay

A proliferation assay was performed on three male cPGC lines from each strain cultured in CSM or OTM media. Cells were seeded into a 96-well plate at a density of 2 × 10³ cells per well (2000 cells/well on Day 0). After that, cell numbers were counted on days 2, 4, 6, 8, and 10. At each time point, 10 µL of cPGCs were collected from each well, stained with Trypan Blue (Thermo Fischer, USA), and counted using a hemocytometer.

Transfection of PGCs

For lipofection, each Bovans cPGC culture, whether in CSM or OTM, was treated with 1 µL of Lipofectamine LTX reagent diluted in 50 µL of Opti-MEM medium (referred to as ‘diluted Lipofectamine’). Concurrently, two µg of DDX4-tdTomato or DAZL-tdTomato plasmids¹¹ were diluted in 50 µL of Opti-MEM medium (referred to as ‘diluted DNA’). The diluted DNA was combined with the diluted Lipofectamine to form a Lipoplex, which was incubated for 30 min. Subsequently, 0.1 × 10⁶ of cPGC lines were counted, pelleted, and resuspended in 100 µL of Opti-MEM medium. The cell suspension was transferred to a 96-well plate, and then 100 µL of Lipoplex was added. Six hours post-transfection, the medium was replaced with fresh CSM or OTM.

For electroporation, Hubbard-derived cPGCs maintained in either CSM or OTM were processed using a NEON electroporation instrument (Neon NxT, Thermo Fischer Scientific, USA), following the protocol outlined in previous work⁷. Briefly, two µg of either DDX4-tdTomato or DAZL-GFP transposon plasmids, along with two µg of piggyBac transposase plasmid, were electroporated by applying 850 V for 50 milliseconds with a single pulse. The expression of tdTomato or GFP in the transfected cPGCs was assessed using fluorescence microscopy 72 h post-transfection for each strain cultured in the respective media.

Immunocytochemistry

Hubbard cPGCs cultured for over 100 days were characterized for the presence of germ cell markers (EMA1, DAZL, DDX4) and a stem cell marker (SSEA1) using immunocytochemistry (detailed methods in⁷). Primary antibodies against EMA1 (mouse IgG; DSHB, USA), DAZL (mouse IgG; Abcam ab34139, UK), DDX4 (mouse IgG; custom made by Biotem, France), and SSEA1 (mouse IgG; DSHB, USA) were employed to detect their corresponding protein markers. FITC-labeled anti-mouse IgM (JIR, USA) and FITC-labeled anti-rabbit IgG (JIR, USA) were used to detect primary antibodies. DNA was counterstained using TO-PRO-3.

Flow cytometry

Expression of EMA1 and SSEA1 was further assessed using specific antibodies and flow cytometry. cPGCs were labeled with primary antibodies EMA1 (1:100; DSHB, USA) and SSEA1- Alexa Fluor 647 (1:125; Santa Cruz, USA). Anti-mouse IgM AF647 (1:500; JIR, USA) was used as a secondary antibody for EMA1-labeled cells. The labeling process consisted of resuspending cPGCs in PBS with 5% FBS, incubating with primary antibodies for 1 h, washing, and incubating with the secondary antibody for 30 min. Control cells were labeled with only the secondary antibody. All centrifugation steps were performed at 1200 rpm for 3 min. The labeled cells were analyzed using a BD flow cytometer (BD Biosciences, USA).

Promoter assay

The activity of germ cell promoters in Bovans cPGCs cultured for extended periods (> 100 days) was assessed using a promoter assay technique. Cells were transfected with plasmids containing the tdTomato reporter gene under the control of DDX4 or DAZL promoters, and the expression of the red fluorescent protein was evaluated under a fluorescence microscope.

RT-PCR gene expression assay

The expression of stem cell-related genes, including core pluripotency genes (NANOG, SOX2, and POU5F3) and pluripotency-associated genes (KLF4, c-MYC, and LIN28A), was evaluated by RT-PCR. Total RNA was isolated from 1 × 10⁵ cPGCs and reverse-transcribed into cDNA with random hexamer using a Thermostable RT kit, following the manufacturer’s instructions (DENAzist Asia, Iran). The transcription of the ACTB gene was used as a control for the reverse transcription step. PCR reactions included 10 µL from 2X Master Mix RED (Ampliqon, Denmark), 1µL of each forward and reverse primers for each gene (Table 2), 2 µL cDNA template (This was equivalent to 2000 ng of input RNA), and 7 µl sterile-deionized water. The PCR steps consisted of an initial denaturation at 94 °C for 3 min, followed by 45 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for specific genes and 55 °C for the control gene for 30 s, extension at 72 °C for 15 s, and a final extension at 72 °C for 5 min.

qPCR gene expression assay

We evaluated the expression levels of NANOG and POU5F3 by RT-qPCR in six established Bovans and Hubbard cPGC lines cultured in two different media. Total RNA was isolated from these cPGC lines using the RNA Isolation Kit (Column RNA Isolation Kit, DENAzist Asia, IRAN for Bovans cPGCs and RNeasy Mini Kit, Qiagen, Germany for Hubbard cPGCs). From 500 ng of input RNA, cDNA was synthesized using the cDNA Synthesis Kit with random hexamer (Thermo Fisher Scientific, USA). qPCR was conducted with 50 ng cDNA in a 20 µL reaction of RealQ Plus 2×Master Mix Green (Ampliqon, Denmark) containing specific primers (Table 2) and an Applied Biosystems Real-Time PCR instrument (Thermo Fisher Scientific, USA). The qPCR temperature steps were the initial denaturation step at 94 °C for 15 min, followed by 45 cycles of denaturation at 94 °C for 30 s, annealing (at 50 °C for NANOG and POU5F3 and 55 °C for ACTB) for 30 s, and extension at 72 °C for 20 s, with a final 10 s extension at 72 °C for signal detection. A melting curve analysis was conducted between 60 °C and 95 °C. Gene expression levels of NANOG and POU5F3 were calculated using the Pfaffl method and ACTB as a calibrator.

PKH26-Red staining of PGCs

The Bovans cPGC line was stained with the PKH26-Red fluorescent cell linker kit (Sigma, USA) for general cell membrane labeling. Briefly, 1 × 10⁶ cPGCs were counted and pelleted. The cells were washed once with a serum-free medium. After centrifugation, the supernatant was cautiously removed without disturbing the cell pellet. Then, 0.5 mL of diluent C reagent was added to the cell pellet and gently pipetted to ensure complete resuspension (2x cell suspension). 2 µL of the PKH26-Red dye solution was added to the 0.5 mL of diluent C and mixed well (2x dye solution). The 2x cell suspension was added to the 2x dye solution, immediately mixed by gentle pipetting, and incubated for 2 min with periodic mixing. The staining procedure was stopped by adding an equal volume (1 mL) of chicken serum, followed by a 1-minute incubation at room temperature. Then, the cells were pelleted at 1200 rpm for 3 min and the supernatant was cautiously discarded. The cell pellet was resuspended in 2 mL of complete medium and transferred to a new sterile conical tube. The cells were washed by pelleting at 1200 rpm for 3 min. To remove the unbound dye, the washing step was repeated two more times with 2 mL of the complete medium. After the final wash, the cell pellet was resuspended in a complete medium for injection into HH 14–16 stage embryos.

Generation and analysis of transgenic Bovans chickens

Male and female Bovans cPGCs cultured in CSM (each 0.1 × 10⁶ cells) were transfected with DDX4-tdTomato transposon and transposase plasmids using Lipofectamine LTX, as described above. Transfection was confirmed by the detection of red fluorescence. Pooled male and female cPGCs were injected into 30 embryonated eggs at the stage of HH 14–16 (around 0.5 × 10⁴ cPGCs per egg).

Only male G0 chickens were raised to puberty. DNA extraction from sperm was conducted using the Animal Tissue DNA Isolation Kit (DENAzist Asia, Iran). Extracted sperm DNA was evaluated for transgene integration using PCR with specific primers (tdTomato) designed for the tdTomato sequence (Table 2). The PCR protocol consisted in an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 15 s, with a final extension at 72 °C for 10 min. The PCR amplicon was 737 bp. To obtain G1 heterozygous chicken, the G0 germline tdTomato-positive roosters were mated with wild-type hens. The same PCR protocol was performed on DNA extracted from the root of the feathers of G1 chickens.

The 737 bp amplicon amplified from DDX4-tdTomato plasmid (as a positive control) and all positive G0 or G1 chickens were recovered from agarose gel using the Gel Extraction Kit (DENAzist Asia, IRAN) and then was digested with the BstXI enzyme (Thermo Fisher, USA) at 55 °C for 3 h. Then digested amplicons were run on a 2% agarose gel.

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• Source: https://www.nature.com/articles/s41598-025-93777-w