Long-term culture of chicken primordial germ cells isolated from embryonic blood and production of germline chimaeric chickens

Accepted Manuscript Title: Long-term culture of chicken primordial germ cells isolated from embryonic blood and production of germline chimaeric chickens Author: Mitsuru Naito Takashi Harumi Takashi Kuwana PII: DOI: Reference:

S0378-4320(14)00384-4 http://dx.doi.org/doi:10.1016/j.anireprosci.2014.12.003 ANIREP 5117

To appear in:

Animal Reproduction Science

Received date: Revised date: Accepted date:

9-6-2014 29-11-2014 1-12-2014

Please cite this article as: Naito, Mitsuru, Harumi, Takashi, Kuwana, Takashi, Long-term culture of chicken primordial germ cells isolated from embryonic blood and production of germline chimaeric chickens.Animal Reproduction Science http://dx.doi.org/10.1016/j.anireprosci.2014.12.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Long-term culture of chicken primordial germ cells isolated from embryonic blood and production of germline chimaeric chickens

Mitsuru Naitoa, Takashi Harumia and Takashi Kuwanab

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International Institute of Avian Conservation Science, P.O. Box 47087, Abu Dhabi, United

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National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan

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Short title: PGC culture and germline chimaeric chickens

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Arab Emirates

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Correspondence should be addressed to: Dr. Mitsuru Naito; National Institute of

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Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan; TEL: +81-29-838-7927; FAX:

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+81-29-838-7408; Email: [email protected]

Highlights

► 1. A novel long-term culture for chicken PGCs especially for female PGCs was developed. ► 2. Feeder cells for culturing PGCs were not derived from xeno-animal cells. ► 3. Frozen-thawed cultured PGCs gave rise to functional gametes in recipient gonads. ► 4.

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PGC culture system is useful for germline manipulation in chickens.

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Cultured PGCs gave rise to viable offspring via germline chimaeric chickens. ► 5. This novel

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ABSTRACT

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Production of germline chimaeric chickens by the transfer of cultured primordial germ cells

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(PGC) is a useful system for germline manipulation. A novel culture system was developed

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for chicken PGC isolated from embryonic blood. The isolated PGC were cultured on feeder

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cells derived from chicken embryonic fibroblast. The cultured PGC formed colonies and they

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proliferated about 300-times during the first 30 days. The cultured PGC retained the ability to migrate to recipient gonads and were also chicken VASA homologue (CVH)-positive. Female

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PGC were present in the mixed-sex PGC populations cultured for more than 90 days and gave rise to viable offspring efficiently via germline chimaeric chickens. Male cultured PGC were transferred to recipient embryos and produced putative chimaeric chickens. The DNA derived from the cultured PGC was detected in the sperm samples of male putative chimaeric

chickens, but no donor derived offspring were obtained. Donor-derived offspring were also obtained from germline chimaeric chickens by the transfer of frozen-thawed cultured PGC. The culture method for PGC developed in the present study is useful for manipulation of the germline in chickens, such as preservation of genetic resources and gene transfer.

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Key words: chicken, culture, embryo, germline chimaera, primordial germ cell

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1. Introduction

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Primordial germ cells (PGC) are progenitor cells of ova and spermatozoa. In chickens,

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PGC appear in the central part of the area pellucida at Stage X (freshly laid egg stage;

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Eyal-Giladi and Kochav, 1976), migrate to the germinal crescent region at Stage 4

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(Hamburger and Hamilton, 1951), and then enter the developing blood vascular system. These

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cells subsequently circulate temporarily in the bloodstream during Stages 13 to 15 and then

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migrate to the germinal ridges (presumptive gonads) and differentiate into male or female

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gametes (Kuwana, 1993). Thus, PGC are the most attractive cells for germline manipulation

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in chickens. Germline chimaeric chickens can be produced by the transfer of PGC, and the transferred PGC can give rise to functional gametes in the gonads of germline chimaeric

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chickens (Tajima et al., 1993; Naito et al., 1994ab, 1998a, 1999). This manipulation technique for PGC can be applied for preservation of avian genetic resources (Naito et al., 1994b; Naito, 2003, 2015; Tajima et al., 1998; Glover and McGrew, 2012), propagation of endangered avian species (Kang et al., 2008; Werney et al., 2010; Liu et al., 2012; van de Lavoir et al., 2012),

and introduction of exogenous DNA into the chicken germline (Naito et al., 1998b, 2007, 2012; van de Lavoir et al., 2006; Macdonald et al., 2012; Park and Han, 2012). The number of PGC that can be obtained from one embryo at the blood circulation stage (Stages 14 to 15) is limited (Naito et al., 1994a; Tajima et al., 1999). To make effective use of a small number of PGC, it is essential to increase the number by culturing these cells in vitro

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and this culture technique for the PGC leads to wide range of applications on germline

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manipulation. The first successful long-term culture of PGC was reported by van de Lavoir et

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al., (2006). In this previous research, PGC were obtained from embryonic blood and were

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cultured on mouse STO or buffalo rat liver (BRL) feeder cells with basic fibroblast growth

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factor (bFGF) and stem cell factor (SCF) supplementation. The PGC were cultured for more

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than 35 days and successfully migrated to the germinal ridges after transfer into the

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bloodstream of recipient embryos. These cells also differentiated into functional gametes in

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recipient gonads. The effectiveness of this culture system for PGC was confirmed by

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Macdonald et al. (2010) and Miyahara et al. (2014). Choi et al. (2010) developed a feeder-free

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culture system for PGC and found that bFGF is one of the important factors for proliferation of PGC and also for maintaining the undifferentiated state of these cells. Furthermore, Park

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and Han (2012) modified the culture system for PGC using mouse fibroblast feeder cells and produced germline chimaeric chickens with more than a 95% germline transmission rate of donor PGC. Because those culture systems use xeno-animal cells as feeder cells, the use of this system may enhance the risk of a cross-transfer of animal pathogens from other animal

cells. It is, therefore, recommended to use chicken cells as feeder cells for culturing chicken PGC. However, long-term culture of chicken PGC isolated from embryonic blood using feeder cells derived from chicken embryos has not been successful. Moreover, because long-term culture of chicken PGC isolated from embryonic blood is only successful in males (van de Lavoir et al., 2006; Macdonald et al., 2010; Park and Han, 2012; Miyahara et al.,

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2014), development of a novel culture method for female PGC is also required.

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Attempts have occurred in developing a PGC culture system using feeder cells derived

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from chicken embryos. The cultured PGC successfully proliferated in vitro, but it was

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difficult to maintain the undifferentiated state of the PGC even in the presence of leukemia

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inhibitory factor (LIF), bFGF and SCF (Naito et al., 2010). The majority of the cultured PGC

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failed to migrate to the germinal ridges after transfer into the bloodstream of recipient

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embryos and only a small proportion of cultured PGC entered the germline and differentiated

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into the functional gametes. To overcome the difficulty of maintaining the undifferentiated

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state of cultured PGC, a novel PGC transfer method has been developed (Naito et al., 2011,

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2012). The cultured PGC, which failed to migrate to the germinal ridges after transfer into the bloodstream of recipient embryos, were transferred to the coelomic epithelium, corresponding

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to the presumptive gonadal region of recipient embryos at Stages 17 to 19. The transferred PGC successfully migrated into the gonads of recipient embryos. This PGC transfer method is effective for cultured PGC which have lost the migratory ability, but sophisticated techniques are required, especially on placing the cultured PGC into the appropriate site of the coelomic

epithelium of recipient embryos. In the present study, a novel culture system was developed for chicken PGC that can proliferate and maintain the undifferentiated state for a long term on feeder cells derived from chicken embryos. The cultured PGC were successfully incorporated into the germline after transfer into the bloodstream of recipient embryos, and differentiated into functional gametes

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in the gonads of germline chimaeric chickens.

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2. Materials and methods

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2.1 Fertilised eggs and animal care

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Fertilised eggs of White Leghorn (WL) and Barred Plymouth Rock (BPR) chickens were

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obtained by artificial insemination. The WL and BPR populations are maintained at the

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National Institute of Livestock and Grassland Science. All animals received humane care as

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outlined in the Guide for the Care and Use of Experimental Animals (National Institute of

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Agrobiological Sciences, Animal Care Committee), and as specifically approved for this

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study (H18-028-1).

2.2 Preparation of donor PGC

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Fertilised eggs of BPR were incubated at 38℃ for about 53 h in a forced-air incubator

(P-008B, Showa Furanki, Saitama, Japan). Blood was collected from the dorsal aorta of embryos at Stages 13 to 15 using a fine glass micropipette. The blood was pooled and dispersed in 400 µl KAv-1 medium (Kuwana et al., 1996) containing 5% foetal bovine serum

(HyClone, Thermo Scientific, South Logan, UT, USA) and 5% chicken serum (Japan Biotest, Tokyo, Japan). The PGC were concentrated by use of the Nycodenz density gradient centrifugation method (Zhao and Kuwana, 2003; Naito et al., 2004). Briefly, 5 ml of 11% Nycodenz (1002424, Axis-Shield, Oslo, Norway) solution was placed in a 50 ml tube (352070, Becton Dickinson, Franklin Lakes, NJ, USA), and 5 ml of 5.5% Nycodenz solution and

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subsequently 400 µl of blood solution were overlaid. The tube was centrifuged at 400 g for 15

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min, and 10 ml of the PGC-rich solution was recovered from the top and washed with KAv-1

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medium.

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2.3 Preparation of feeder cells

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Fertilised eggs of the WL population were incubated for about 57 hours at 38 ℃. When

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the embryos reached Stages 15 to 16, the embryonic body was isolated from the yolk and

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washed with Dulbecco’s phosphate-buffered saline without Ca2+ and Mg2+ (DPBS(-))

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(28-103-05 FN, Dainippon Sumitomo Pharma, Osaka, Japan). The head and heart were

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removed from the embryonic body, cut into small pieces, and cultured in the cell culture flask

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(353813, Becton Dickinson, Franklin Lakes, NJ, USA) after being dispersed in KAv-1 medium. Passages were performed when the fibroblast cells became nearly confluent. The

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cultured fibroblast cells were treated with mitomycin C (20898-21, Nacalai Tesque, Kyoto, Japan), then washed with DPBS(-) three times and the fibroblast cells were collected by trypsin treatment (T4049, Sigma, St. Louise, MO, USA) for 5 minutes at room temperature (25 ℃ ). The collected fibroblast cells were dispersed in fresh KAv-1 medium at a

concentration of 2.5 × 105 cells/ml and seeded on a 4-well plate (176740, Nunc, Roskilde, Denmark) treated with 0.1% gelatin solution (ES-006-B, Millipore, Billerica, MA, USA). 2.4 PGC culture in vitro The PGC (about 3,000) were dispersed in 600 µl of culture medium for PGC and placed on feeder cells. The PGC culture medium used was KAv-1, supplemented with 10 ng/ml

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human bFGF (060-04543, Wako Pure Chemicals, Osaka, Japan), 2% chick embryo extract

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(CEE), and 15% knockout serum replacement (KSR, 10828-028, Invitrogen, Grand Island,

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NY, USA). Chick embryo extract was prepared as follows. The WL embryos that were

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incubated for 5 days were isolated from the yolk, washed with DPBS(-), and each embryo

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was placed in a homogenising tube (Lysing Matrix A, MP-Biomedicals, Solon, OH, USA)

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with 800 µl KAv-1 medium. The tubes were shook vigorously using FastPrep instrument

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(FP100A, MP-Biomedicals, Solon, OH, USA), then centrifuged and supernatants were

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collected. The chick embryo extract solution was filtered (0.45 µm, SLHV033RS, Millex-HV,

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Millipore, Billerica, MA, USA) and stored at -20 ℃ until use. For passage, the cultured PGC

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were isolated from feeder cells by gentle pipetting and then dissociated by trypsin treatment (T4049, Sigma, St. Louis, MO, USA) for 5 minutes at room temperature. After washing with

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fresh KAv-1 medium, the cultured PGC were suspended in the culture medium for PGC and placed on freshly prepared feeder cells. 2.5 Freezing and thawing of cultured PGC Cultured colonies of PGC were isolated from feeder cells and the collected PGC were

dissociated by trypsin treatment for 5 minutes at room temperature. After washing with fresh KAv-1 medium, the cultured PGC were suspended in 200 µl of freezing medium (Cell Banker 3, BLC-3, ZENOAQ, Fukushima, Japan) and put in a cryotube (5000-0012, Nalgen, NY, USA) after counting the number of cells by hemocytometer (DHC-N01, C-CHIP, NanoEnTek, Seoul, Korea). The cryotube was placed in a freezer (-80 ℃) and stored for 24 hours. Then

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the cryotube was placed in liquid nitrogen (-196 ℃) for 9 days to 3 months. The cryotube

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was taken out of liquid nitrogen and immediately put into water at 37 ℃. After thawing, the

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cell suspension was washed with fresh KAv-1 medium and dispersed in culture medium and

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placed on freshly prepared feeder cells after counting the number of cells by hemocytometer.

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The viability of the frozen-thawed cultured PGC was determined by Trypan blue exclusion

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method (Freshney, 1987). One drop of the cell suspension and one drop of 0.5% Trypan blue

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solution (29853-34, Nakalai Tesque, Kyoto, Japan) were mixed and incubated for 1 to 2

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minutes at room temperature, and the number of stained and non-stained cells were counted.

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2.6 Sexing of cultured PGC

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Cultured PGC were isolated from feeder cells by gentle pipetting, only colonies were picked up, and then DNA was extracted using DNA extraction kit (SepaGene, Sanko Junyaku,

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Tokyo, Japan) according to the manufacturer’s instructions. The extracted DNA was dissolved in distilled water at a concentration of 100 ng/µl, and PCR analysis was then performed to detect the presence of the W chromosome-specific repeating sequences. The PCR analysis for sexing was performed using a programmable thermal controller (Model 9700; Perkin Elmer,

U.S.A.). The PCR reaction mixture was prepared using Takara Ex Taq kit (PR001, Takara Bio Inc., Shiga, Japan), and the reaction was performed in 25 µl reactions containing 50 ng genomic DNA, 0.5 µM primers, 0.2 mM dNTPs, and 0.5U DNA polymerase. The sequences of the primers for detecting W chromosome-specific repeating sequences were: 5’-CCC AAA TAT AAC ACG CTT CAC T-3’, and 5’-GAA ATG AAT TAT TTT CTG GCG AC-3’ (Clinton

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et al., 2001). Control PCR reactions were conducted in the same sample (a single tube assay)

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to ensure the presence of genomic DNA using primers: 5’-AGC TCT TTC TCG ATT CCG

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TG-3’, and 5’-GGG TAG ACA CAA GCT GAG CC-3’ designed to amplify the chicken 18S

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ribosomal gene (Clinton et al., 2001). After an initial denaturation step of 94 ℃ for 2

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minutes, 25 cycles of amplification were performed; DNA was denatured at 94 ℃ for 30

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seconds, annealed at 56 ℃ for 30 seconds, and extended at 72 ℃ for 30 seconds. The

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samples were then incubated at 72 ℃ for 5 minutes. After amplification, 5 µl of the reaction

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product was separated on a 2% agarose gel and visualised under UV irradiation after ethidium

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bromide staining. A band of 415 bp was detected in females but not in males, and a band of

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256 bp was detected for the control reaction. 2.7 Immunohistochemical analysis of cultured PGC

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Germline cell characteristics of the cultured PGC were identified by immunostaining.

The cultured cells were fixed with 4% paraformaldehyde (163-20145, Wako Pure Chemical, Osaka, Japan) for 1 hour. After washing with DPBS(-), blocking was performed with Blocking One (03953-95, Nacalai Tesque, Kyoto, Japan) for 1 hour. The cells were then

incubated with chicken VASA homologue (CVH) antibodies (1:4,000 dilution, Tsunekawa et al., 2000) for 1 hour. After a second washing with DPBS(-), the cells were incubated with alkaline phosphatase-labelled goat anti-rabbit immunoglobulin (1:200 dilution, SAB1005, Open Biosystems, Huntsville, AL, USA) for 30 min. The cells were then washed with DPBS(-), incubated with 5-bromo-4-chloro-3-indoxyl phosphate/nitro blue tetrazolium

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chloride substrate (K0598, Dako Cytomation, Glostrup, Denmark) for several minutes, and

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microscope (DMIRE2, Leica Microsystems, Tokyo, Japan).

Sigma,

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2.8 Sterilisation of recipient embryos Busulfan

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then washed with distilled water. The treated cells were observed under an inverted

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N,N-Dimethylformamide (08900-12, Nacalai Tesque, Kyoto, Japan) at a concentration of 80

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mg/ml, diluted 160-fold with PBS(-), and emulsified with sesame oil (25620-65, Nacalai

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Tesque, Kyoto, Japan) as described previously (Nakamura et al., 2009, 2010) using SPG

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membrane filter (SPG pumping connector, SPG Techno, Miyazaki, Japan). Freshly laid

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fertilised eggs at Stage X from the WL population were broken, the albumen capsule removed, and the yolk put in a glass vessel placing the blastoderm on top of the yolk. The prepared

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busulfan solution of 5 µl was injected into the yolk immediately under the blastoderm layer using a glass micropipette. The yolk was then placed in a small host eggshell and filled with thin albumen. The opening of the eggshell was covered with cling film and secured by the plastic rings and elastic bands (System II, Perry, 1988; Naito et al., 1990). The reconstituted

eggs were incubated for 2.5 days until the embryo reaches Stages 14 to 16 in a forced air incubator. 2.9 Analysis of migratory ability of cultured PGC to recipient gonads Cultured PGC were isolated from the feeder cells by gentle pipetting and then dissociated by trypsin treatment for 3 minutes at room temperature. The dissociated cultured

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PGC were labelled with fluorescent dye (PKH26, Sigma, St. Louis, MO, USA) according to

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the manufacturer’s instructions. The fluorescent labelled PGC (200 PGC) were transferred to

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the Stage X blastoderm after breaking the egg (Naito et al., 1991), and the embryos (yolks)

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were transferred to small host eggshells (System II) and cultured for 2.5 days. The embryos

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were again transferred to large host eggshells (System III, Perry, 1988; Naito et al., 1990) and

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cultured for a further 5 days. The fluorescent labelled PGC (200 PGC) were also transferred

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into the bloodstream of recipient embryos (WL) at Stages 14 to 16 of which endogenous PGC

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were partially sterilised with busulfan treatment at Stage X. The embryos were transferred to

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large host eggshells and cultured for a further 5 days. Frozen-thawed cultured PGC (500

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PGC) were labelled with fluorescent dye (PKH67, Sigma, St. Louise, MO, USA) and transferred to the partially sterilised recipient embryos at Stages 14 to 16. The embryos were

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then cultured for a further 5 days. The cultured embryos were isolated from the yolk, washed with DPBS(-), and the gonads were exposed and removed from the embryos. The presence of the fluorescent labelled cells in the gonads was examined under a fluorescent microscope (M205 FA, Leica Microsystems, Tokyo, Japan).

2.10 Detection of donor-derived cells in recipient gonads The PGC cultured for 90 to 97 days were isolated from the feeder cells, dissociated by trypsin treatment, and 200 PGC were transferred to the bloodstream of partially sterilised recipient embryos (WL). The embryos were cultured for a further 17 days in large host eggshells. The gonads and a small portion of liver (as control) were subsequently isolated

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from the embryos. The DNA was extracted from the gonads and livers, and the DNA was

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dissolved in distilled water at a concentration of 100 ng/µl. The PCR analysis was then

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performed to detect the presence of donor-derived cells (BPR). Detection of WL- and

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BPR-specific sequences in the PMEL17 gene (Kerje et al., 2004) was performed by the

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method of Choi et al. (2007) with some modifications (Naito et al., 2012). The primer

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sequences were 5′-CTG CCT CAA CGT CTC GTT GGC-3′ and 5′-AGC AGC GGC GAT

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GAG CGG TG-3′ for detecting WL, and 5′-CTG CCT CAA CGT CTC GTT GGC-3′ and

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5′-AGC AGC GGC GAT GAG CAG CA-3′ for detecting BPR. The PCR mixture was

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prepared using the Takara PrimeSTAR GXL kit (R050A, Takara Bio Inc. Tokyo, Japan), and

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the reaction was performed in 25 µl reactions containing 50 ng genomic DNA, 0.5 µM primers, 0.2 mM dNTPs, and 0.25U DNA polymerase. After an initial denaturation step of

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94 ℃ for 5 min, 40 cycles were conducted; 30 seconds of denaturation at 94 ℃, 30 seconds of annealing and extension at 72 ℃, and a final 5 minute extension at 72 ℃. After amplification, the PCR products were separated on a 2% agarose gel with the bands (WL: 222 bp, BPR: 213 bp) visualised under UV light after ethidium bromide staining.

2.11 Production of germline chimaeric chickens and test mating There were 200 to 500 PGC (BPR) cultured for 15 to 120 days, frozen-thawed (70 to 126 days culture, 9 to 16 days in the frozen state and 25 to 44 days of culture) or freshly collected (control) that were transferred to the bloodstream of partially sterilised recipient embryos (WL) at Stages 14 to 16. The embryos were then transferred to the large host eggshells

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(System III) and cultured for a further 18 days until hatching. The hatched chicks were raised

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until sexual maturity and both male and female putative chimaeric chickens were mated with

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BPR by artificial insemination. The fertilised eggs obtained were incubated for 16 days or

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until hatching, and the feather colour of the developing embryos was examined. The WL is

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homozygous dominant (I/I) at the autosomal pigment inhibitor gene and the chick feathers are

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white, while the BPR is homozygous recessive (i/i) and their feathers are black. Black

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offspring (i/i) indicate that the embryos were derived from the donor BPR PGC transferred,

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while the white offspring (I/i) derivation from the endogenous WL PGC was confirmed.

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Concurrently with the test mating, sperm samples were collected from male putative

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chimaeric chickens and analysed for the presence of transferred donor PGC-derived

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spermatozoa (BPR) by PCR after extracting DNA.

3. Results 3.1 Proliferation of cultured PGC in vitro Cultured PGC started to proliferate slowly at an initial stage of in vitro culture. At 10 to

14 days of culture, colonies of PGC became visible (Fig. 1A). These colonies attached loosely to the feeder layer and were easily isolated by gentle pipetting. The collected PGC were dissociated by trypsin treatment and the morphology was observed. Most of these cultured PGC maintained the typical PGC morphology, large cells with an eccentrically positioned nucleus and a considerable amount of lipid droplets in the cytoplasm (Fig. 1B). After passages,

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many PGC proliferated and formed colonies (Fig. 1C), and most of the cultured PGC also

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maintained the typical PGC morphology (Fig. 1D). After several passages, many PGC

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continued to proliferate and formed colonies (Fig. 1E, 1F). Proliferation of cultured PGC for

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the first 30 days is shown in Fig. 2. About 3,000 PGC proliferated 40-times during the first 10

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days of culture, and then the PGC proliferated rapidly and reached approximately 1,000,000

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cells at 30 days of culture.

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3.2 Recovery rate and viability of frozen-thawed cultured PGC

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The cultured PGC were successfully frozen and thawed. Numbers of cultured PGC were

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counted before freezing and after thawing. The recovery rate of the cultured PGC after

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freezing and thawing was 42.3% (188,000/444,000), and the viability of the frozen-thawed cultured PGC was 92.0% (355/387). The frozen-thawed cultured PGC proliferated and

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formed colonies after culturing on feeder cells (Fig. 1G, 1H). 3.3 Presence of female cells in cultured PGC populations Initial populations of PGC were mixed-sex populations. The PGC colonies cultured for 90 to 97 days were carefully isolated from the feeder layer, then analysed for the presence of

female cells after extracting DNA from the colonies. As a result, all the populations of PGC that were analysed contained the W-chromosome specific repeating sequences (female cells) as shown in Fig. 3. 3.4 Immunohistochemical analysis of cultured PGC The PGC proliferated and usually formed colonies. These PGC were fixed and stained

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with anti-CVH antibody at Day 90 of culture. Most of the colonies of PGC were

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CVH-positive (Fig. 4A), but some PGC colonies were CVH-negative (Fig. 4B). The

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CVH-positive PGC colonies attached loosely to the feeder layer while CVH-negative colonies

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of PGC attached firmly to the feeder layer. No apparent differences were observed in the

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shape and morphology of the cultured PGC between CVH-positive and CVH-negative

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colonies.

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3.5 Migration of cultured PGC to recipient gonads

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When PGC cultured for 31 days with fluorescent labelling were transferred to the Stage

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X blastoderm of recipient embryos, these cells entered the bloodstream and migrated to the

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gonads (6/6, 100%; Fig. 5A, 5B). No feather chimaeras were observed (0/12) in the embryos at Day 17 of incubation. When PGC cultured for 31 days with fluorescent labelling were

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transferred to the bloodstream of recipient embryos, these cells migrated to the gonads (11/11, 100%; Fig. 5C, 5D). The fluorescent labelled cells which migrated to the recipient gonads and dispersed throughout the gonads in both cases, and the efficiency of migration to the gonads tends to be greater when the cultured PGC were transferred to the bloodstream compared with

the transfer to the Stage X blastoderm (Fig. 5B, 5D). The fluorescent labelled frozen-thawed cultured PGC were also transferred to the bloodstream of recipient embryos, and these cells migrated to the gonads of recipient embryos (Fig. 5E, 5F). Migration of cultured PGC to recipient gonads was also confirmed by PCR analysis. PGC cultured for 90 to 97 days were transferred to the bloodstream of recipient embryos and

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the embryos were cultured up to Day 20.5 of incubation. The gonads and a small portion of

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liver were isolated from the embryos and analysed for the presence of the transferred

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donor-derived cells by PCR. A part of the PCR analysis is shown in Fig. 6. The presence of

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cultured PGC-derived cells were detected in all the gonads that were analysed (50/50, 100%),

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but not in livers (0/50, 0%), as shown in Lanes 5 to 10 of Fig. 6.

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3.6 Production of germline chimaeric chickens and test mating

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Cultured PGC, frozen-thawed cultured PGC and freshly collected PGC were transferred

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to the partially sterilised recipient embryos. The rates of hatching of the embryos transferred

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the cultured PGC, frozen-thawed cultured PGC and freshly collected PGC were 28.0%

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(121/432), 30.6% (11/36) and 27.8% (20/72), respectively, and 57, 5 and 8 putative chimaeric chickens matured sexually and test mating was performed (Table 1). The results of the test

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mating are shown in Table 2. When freshly collected PGC were transferred to the recipient embryos, all the putative chimaeric chickens proved to be germline chimaeras. The germline transmission rates of donor-derived gametes were 82.1% to 100.0% in males and 87.7% to 90.8% in females. When cultured PGC were transferred to the recipient embryos, 46.7%

(14/30) of the female putative chimaeric chickens proved to be germline chimaeras. The germline transmission rates of donor-derived gametes were 2.9% to 100.0%. On the contrary, no donor-derived offspring were obtained so far from the 27 male putative chimaeric chickens, although donor-PGC derived DNA was detected in the sperm samples obtained from the male putative germline chimaeric chickens (20/27, 74.1%; Fig. 7). When frozen-thawed cultured

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PGC were transferred to the recipient embryos, one female germline chimaeric chicken was

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detected of five putative chimaeric chickens that were tested. The germline transmission rate

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of donor-derived gametes was 21.1%. A part of the hatched chicks derived from the cultured

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PGC and the frozen-thawed cultured PGC are shown in Fig. 8A and 8B, respectively.

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4. Discussion

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In the present study, chicken PGC isolated from embryonic blood were successfully

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cultured in vitro using feeder cells derived from chicken embryos. The cultured PGC formed

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colonies and proliferated rapidly. These cells attached loosely to the feeder layer and were

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easily detached by gentle pipetting. The proliferation rate of the cultured PGC was more than 300-times during the first 30 days of culture. Morphology of the cultured PGC after several

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passages was similar to the freshly collected PGC. Most of the colonies of PGC cultured for 90 days were CVH-positive, suggesting that the cultured PGC retained the characteristics of germline cells. Some colonies of PGC attached firmly to the feeder layer and were CVH-negative, indicating that these CVH-negative PGC were differentiated. The cultured

PGC have also retained the migratory ability to the germinal ridges after transfer to the Stage X blastoderm or to the bloodstream of recipient embryos. Migration of the cultured PGC to the recipient gonads was confirmed by the presence of the fluorescent-labelled cells in the recipient gonads and also PCR analysis of DNA extracted from the gonads of 19.5-day incubated embryos. Finally, generation of donor-derived offspring from the female chimaeric

PT

chickens produced by the transfer of cultured PGC indicates that the cultured PGC have an

RI

ability to differentiate into functional gametes in females, but not in males. Thus, by using the

SC

culture method developed in the present study, PGC can be maintained and proliferated

U

without using STO or BRL feeder cells and BRL-conditioned medium as has been reported by

N

van de Lavoir et al. (2006).

M

A

When PGC isolated from embryonic blood were cultured in vitro, PGC colonies became

D

visible at 10 to 14 days of culture. By observing the fate of cultured PGC, it was found that

TE

not all the PGC proliferated and formed colonies. The PGC circulating in the bloodstream

EP

most likely were not a homologous population, and some of these cells have the capacity to

CC

proliferate actively in vitro. van de Lavoir et al. (2006) suggested that the starting population of PGC was heterogeneous and that a small portion of the population of PGC regularly

A

differentiates. It is also reported that the total population of PGC are composed of subpopulations that have a greater ability to develop into EG (embryonic germ) cells and these subpopulations are relatively undifferentiated (Matsui and Tokitake, 2009). It is, thus, probable that the population of PGC in chickens isolated from embryonic blood is a mixture

of heterogeneous PGC and that some part of the population can proliferate and maintain the undifferentiated state for a long term in vitro. The LIF, bFGF and SCF are important for proliferation and survival of PGC (van de Lavoir et al., 2006). By supplementing bFGF to the culture medium, PGC could be maintained in vitro without differentiation or de-differentiation in feeder free conditions,

PT

suggesting that bFGF is an essential factor for in vitro PGC proliferation (Choi et al., 2010).

RI

The bFGF stimulated PGC proliferation, but not to the extent to maintain the undifferentiated

SC

state of PGC (Naito et al., 2010). In a preliminary study, effects of CEE and KSR on PGC

U

proliferation was examined. The CEE was obtained from 5-day incubated embryos in the

N

present study, because PGC at this stage maintain an undifferentiated state and retain

M

A

migratory ability. When CEE was added to the culture medium, proliferation of PGC was

D

stimulated but the morphology of the PGC was changed similar to somatic cells. Next, CEE

TE

and KSR were added to the culture medium, and PGC actively proliferated and also

EP

maintained the PGC-specific morphology. The CEE most likely stimulated proliferation of

CC

PGC and KSR maintained the undifferentiated state of PGC in vitro. The mechanism of KSR for maintaining the undifferentiated state of PGC is unknown because the contents of KSR

A

have not been disclosed. Concerning male and female lines of PGC, van de Lavoir et al. (2006) reported that the two female lines of PGC that were established could not be maintained beyond 109 and 77 days, although male lines of PGC could be maintained for a long term. It is unknown why

female lines of PGC could not be maintained for the long term compared with male lines of PGC (van de Lavoir et al., 2006; Macdonald et al., 2010; Park and Han, 2013; Miyahara et al., 2014). In the present study, the starting population of PGC was a mixture of male and female. After 3 months of culture, the presence of female PGC in the cultured populations was analysed by detecting the W chromosome-specific repeating sequences. As a result, it was

PT

confirmed that female PGC were present in the cultured populations, and PGC cultured for

RI

112 days could give rise to functional offspring via female germline chimaeric chickens. This

SC

is the first report on successful long-term culture of female PGC in vitro and subsequent

U

generation of viable offspring via germline chimaeric chickens. It is suggested that optimal

N

culture conditions of female PGC are different from male PGC, and the culture conditions

M

A

developed in the present study provide the opportunity to analyse the factors involved in the

D

sex-specific culture conditions of PGC. Although donor-derived DNA (BPR) was detected in

TE

the sperm samples obtained from the putative male chimaeric chickens, no donor-derived

EP

offspring were generated from the male putative chimaeric chickens that were tested.

CC

Differentiation of male PGC into functional spermatozoa was arrested at some developmental stages in the gonads of putative male chimaeric chickens. It is necessary to establish male and

A

female lines of PGC derived from single embryos and to compare the possible culture periods and differentiation ability into functional gametes in both male and female lines to examine the sex-specific culture conditions of PGC. Sterilisation of recipient embryos is important for efficient production of donor-derived

offspring from germline chimaeric chickens. Various attempts have been made to reduce the endogenous PGC, such as UV irradiation (Reynaud, 1976; Aige-Gil and Simkiss, 1991), soft X-ray irradiation (Li et al., 2001; Nakamichi et al., 2006), γ-ray irradiation (Carsience et al., 1993), blood removal containing PGC (Naito et al., 1994a) and busulfan administration (Bresler et al., 1994; Song et al., 2005). Administration of busulfan emulsion into the yolk of

PT

Stage X embryo was effective for sterilising endogenous PGC (Nakamura et al., 2009, 2010).

RI

These cells efficiently replaced endogenous germ cells with donor PGC-derived germ cells. In

SC

the present study, busulfan emulsion was administered to the Stage X blastoderm and freshly

U

collected PGC were transferred to the bloodstream of recipient embryos. Male and female

N

germline chimaeric chickens produced with these treatments generated donor-derived

M

A

offspring with the frequency of more than 80%. Female germline chimaeric chickens

D

produced by the transfer of cultured PGC, however, generated donor-derived offspring with

TE

various frequencies. The frequencies tended to be greater when the culture period for PGC

EP

was short and the frequencies gradually decreased when the period of PGC culture increased.

CC

Most likely a small portion of the PGC in a population occasionally differentiated during the long-term culture period, and as a result the rate of germline-competent PGC in a population

A

would gradually decrease. Freshly collected PGC can be preserved in liquid nitrogen and subsequently give rise to viable offspring via germline chimaeric chickens (Naito et al., 1994b; Tajima et al., 1998). Cultured PGC could also be preserved in liquid nitrogen and subsequently proliferate in

culture and give rise to viable offspring via germline chimaeric chickens as shown in the present study. In conclusion, the culture system for PGC developed in the present study is very useful for preservation and propagation of avian genetic resources. This culture system for PGC is also useful for manipulating the germline in chickens, especially for propagation of

SC

RI

contribute to further development in avian germline manipulation.

PT

endangered avian species and producing transgenic chickens. The present study will

U

Declaration of interest

N

The authors declare that there is no conflict of interest that could be perceived as

TE

Acknowledgements

D

M

A

prejudicing the impartiality of the research reported.

EP

The authors would like to thank the staff of the Poultry Management Section of the

CC

National Institute of Livestock and Grassland Science for taking care of the birds and providing the fertilised eggs. This study was supported by a Grant-in-Aid (No. 20380156)

A

from the Japan Society for the Promotion of Science, Kieikai Research Foundation, and II-ACS Research Fund to MN.

References

Aige-Gil, V., Simkiss, K., 1991. Sterilising embryos for transgenic chimaeras. Brit. Poult. Sci. 32, 427-438. Bresler, M., Behnam, J., Luke, G., Simkiss, K., 1994. Manipulations of germ-cell populations in the gonad of the fowl. Brit. Poult. Sci. 35, 241-247. Carsience, R.S., Clark, M.E., Verrinder Gibbins, A.M., Etches, R.J., 1993 Germline chimeric

PT

chickens from dispersed donor blastodermal cells and compromised recipient embryos.

RI

Development 117, 669-675.

SC

Choi, J.W., Lee, E.Y., Shin, J.H., Zheng, Y., Cho, B.W., Kim, J.K. Han, J.Y., 2007.

U

Identification of breed-specific DNA polymorphisms for a simple and unambiguous

N

screening system in germline chimeric chickens. J. Exp. Zool. 307A, 241-248.

M

A

Choi, J.W., Kim, S., Kim, T.M., Kim, Y.M., Seo, H.W., Park, T.S., Jeong, J.W., Song, G., Han,

D

J.Y., 2010. Basic fibroblast growth factor activates Mek/Erk cell signaling pathway and

TE

stimulates the proliferation of chicken primordial germ cells. PLoS ONE 5(9) e12968.

EP

doi:10.1371/journal.pone.0012968.

CC

Clinton, M., Haines, L., Belloir, B., McBride, D., 2001. Sexing chick embryos: a rapid and simple protocol. Brit. Poult. Sci. 42, 134-138.

A

Eyal-Giladi, H., Kochav, S., 1976. 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, 321-337. Freshney, R.I., 1987. Culture of Animal Cells, Edn 2, pp 245-256. New York: Alan R Liss.

Glover, J.D., McGrew, M.J., 2012. Primordial germ cell technologies for avian germplasm cryopreservation and investigating germ cell development J. Poult. Sci. 49, 155-162. Hamburger, V., Hamilton, H.L., 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 8, 49-92. Kang, S.J., Choi, J.W., Kim, S.Y., Park, K.., Kim, T.M., Lee, Y.M., Kim, H., Lim, J.M., Han,

PT

J.Y., 2008. Reproduction of wild birds via interspecies germ cell transplantation. Biol.

RI

Reprod. 79, 931-937.

SC

Kerje, S., Sharma, P., Gunnarsson, U., Kim, H., Bagchi, S., Fredriksson, R., Schutz, K.,

U

Jensen, P., von Heijine, G., Okimoto, R., Andersson, L., 2004. The Dominant white, Dun

N

and Smoky color variants in chicken are associated with insertion/deletion

M

A

polymorphisms in the PMEL17 gene. Genetics 168, 1507-1518.

D

Kuwana, T., 1993. Migration of avian primordial germ cells toward the gonadal anlage. Dev.

TE

Growth Differ. 35, 237-243.

EP

Kuwana, T., Hashimoto, K., Nakanishi, A., Yasuda, Y., Tajima, A., Naito, M., 1996.

CC

Long-term culture of avian embryonic cells in vitro. Int. J. Dev. Biol. 40, 1061-1064. Li, H.C., Kagami, H., Matsui, K., Ono, T., 2001. Restriction of proliferation of primordial

A

germ cells by the irradiation of Japanese quail embryos with soft X-rays. Com. Biochem. Physiol. Part A 130, 133-140.

Liu, C., Khazanehdari, K.A., Baskar, V., Saleem, S., Kinne, J., Wernery, U., Chang, I.K., 2012. Production of chicken progeny (Gallus gallus domesticus) from interspecies

germline chimeric duck (Anas domesticus) by primordial germ cell transfer. Biol. Reprod. 86, 101-108. Macdonald, J., Glover, J.D., Taylor, L., Sang, H.M., McGrew, M.J., 2010. Characterisation and germline transmission of cultured avian primordial germ cells. PLoS ONE 5(11) e15518. doi:10.1371/journal.pone.0015518.

PT

Macdonald, J., Taylor, .L, Sherman, A., Kawakami, K., Takahashi, Y., Sang, H.M., McGrew,

RI

M.J. 2012. Efficient genetic modification and germ-line transmission of primordial germ

SC

cells using piggyBac and Tol2 transposons. Proc. Nat. Acad. Sci. USA 109(23),

U

E1466–E1472.

N

Matsui, Y., Tokitake, Y., 2009. Primordial germ cells contain subpopulations that have greater

M

A

ability to develop into pluripotential stem cells. Dev. Growth Differ. 51, 657-667.

D

Miyahara, D., Mori, T., Makino, R., Nakamura, Y., Oishi, I., Ono, T., Nirasawa, K., Tagami,

TE

T., Kagami, H., 2014. Culture conditions for maintain propagation, long-term survival

EP

and germline transmission of chicken primordial germ cell-like cells. J. Poult. Sci. 51,

CC

87-95.

Naito, M., 2003. Genetic manipulation in chickens. World’s Poult. Sci. J. 59, 375-385.

A

Naito M., 2015. Embryo manipulation in chickens. J. Poult. Sci. 52, 7-14. Naito, M., Nirasawa, K., Oishi, T., 1990. Development in culture of the chick embryo from fertilized ovum to hatching. J. Exp. Zool. 254, 322-326. Naito, M., Watanabe, M., Kinutani, M., Nirasawa, K., Oishi, T., 1991. Production of

quail-chick chimaeras by blastoderm cell transfer. Brit. Poult. Sci. 32, 79-86. Naito, M., Tajima, A., Yasuda, Y., Kuwana, T., 1994a. Production of germline chimeric chickens, with high transmission rate of donor-derived gametes, produced by transfer of primordial germ cells. Mol. Reprod. Dev. 39, 153-161. Naito, M., Tajima, A., Tagami, T., Yasuda, Y., Kuwana, T., 1994b, Preservation of chick

PT

primordial germ cells in liquid nitrogen and subsequent production of viable offspring. J.

RI

Reprod. Fert. 102, 321-325.

SC

Naito, M., Tajima, A., Yasuda, Y., Kuwana, T., 1998a. Donor primordial germ cell-derived

U

offspring from recipient germline chimaeric chickens: absence of long term immune

N

rejection and effects on sex ratios. Brit. Poult. Sci. 39, 20-23.

M

A

Naito, M., Sakurai, M., Kuwana, T., 1998b. Expression of exogenous DNA in the gonads of

D

chimaeric chicken embryos produced by transfer of primordial germ cells transfected in

TE

vitro and subsequent fate of the introduced DNA. J. Reprod. Fert. 113, 137-143.

EP

Naito, M., Matsubara, Y., Harumi, T., Tagami, T., Kagami, H., Sakurai, M., Kuwana, T., 1999.

CC

Differentiation of donor primordial germ cells into functional gametes in the gonads of mixed-sex germline chimaeric chickens produced by transfer of primordial germ cells

A

isolated from embryonic blood. J. Reprod. Fert. 117, 291-298.

Naito, M., Sano, A., Harumi, T., Matsubara, Y., Kuwana, T., 2004. Migration of primordial germ cells isolated from embryonic blood into the gonads after transfer to stage X blastoderms and detection of germline chimaerism by PCR. Brit. Poult. Sci. 45, 762-768.

Naito, M., Minematsu, T., Harumi, T., Kuwana, T., 2007. Intense expression of GFP gene in gonads of chicken embryos by transfecting circulating primordial germ cells in vitro and in vivo. J. Poult. Sci. 44, 416-425. Naito, M., Harumi, T., Kuwana, T., 2010. Long term in vitro culture of chicken primordial germ cells isolated from embryonic blood and incorporation into germline of recipient

PT

embryo. J. Poult. Sci. 47, 57-64.

RI

Naito, M., Harumi, T., Kuwana, T., 2011. In vitro culture of testicular and ovarian gonocytes

SC

obtained from 19-day incubated chicken embryos and subsequent colonization into

U

gonads of recipient embryos. J. Poult. Sci. 48, 112-118.

N

Naito, M., Harumi, T., Kuwana, T., 2012. Expression of GFP gene in cultured PGCs isolated

M

A

from embryonic blood and incorporation into gonads of recipient embryos. J. Poult. Sci.

D

49, 116-123.

TE

Nakamichi, H., Sano, A., Harumi, T., Matsubara, Y., Tajima, A., Kosugiyama, M., Naito, M.,

EP

2006. Effects of soft X-ray irradiation to stage X blastoderm on restriction of

CC

proliferation of primordial germ cells in early chicken embryos. J. Poult. Sci. 43, 394-400.

A

Nakamura, Y., Usui, F., Atsumi, Y., Otomo, A., Teshima, A., Ono, T., Takeda, K., Nirasawa, K., Kagami, H., Tagami, T., 2009, Effects of busulfan sustained-release emulsion on depletion and repopulation of primordial germ cells in early chicken embryos J. Poult. Sci. 46, 127-135.

Nakamura, Y., Usui, F., Ono, T., Takeda, K., Nirasawa, K., Kagami, H., Tagami, T., 2010. Germline replacement by transfer of primordial germ cells into partially sterilized embryos in the chicken. Biol. Reprod. 83, 130-137. Park, T.S., Han, J.Y., 2012. piggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens. Proc. Nat. Acad. Sci. USA 109, 9337–9341.

PT

Park, T.S., Han, J.Y., 2013. Conservation of migration and differentiation circuits in

RI

primordial germ cells between avian species. J. Reprod. Dev. 59, 252-257.

SC

Perry, M.M., 1988. A complete culture system for the chick embryo. Nature 331, 70-72.

U

Raynaud, G., 1976 Reproductive capacity and offspring of chickens submitted to transfer of

N

primordial germ cells during embryonic life. Roux’s Arch. Dev. Biol. 179, 85-110.

M

A

Song, Y., D’Costa, S., Pardue, S.L., Petitte, J.N., 2005. Production of germline chimeric

D

chickens following the administration of a busulfan emulsion. Mol. Reprod. Dev. 70,

TE

438-444.

EP

Tajima, A., Naito, M., Yasuda, Y., Kuwana, T., 1993. Production of germ line chimera by

CC

transfer of primordial germ cells in domestic chicken (Gallus domesticus). Theriogenology 40, 509-519.

A

Tajima, A., Naito, M., Yasuda, Y., Kuwana, T., 1998. Production of germ-line chimeras by transfer of cryopreserved gonadal primordial germ cells (gPGCs) in chicken. J. Exp. Zool. 280, 265-267.

Tajima, A., Hayashi, H., Kamizumi, A., Ogura, J., Kuwana, T., Chikamune, T., 1999. Study on the concentration of circulating primordial germ cells (cPGCs) in early chick embryos. J. Exp. Zool. 284, 759-64. Tsunekawa, N., Naito, M., Sakai, Y., Nishida, T., Noce, T., 2000. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development 127,

PT

2741-2750.

RI

van de Lavoir, M.C., Diamond, J.H., Leighton, P.A., Mather-Love, C., Heyer, B.S., Bradshaw,

SC

R., Kerchner, A., Hooi, L.T., Gessaro, T.M., Swanberg, S.E., Delany, M.E. Etches, R.J.,

U

2006. Germline transmission of genetically modified primordial germ cells. Nature 441,

N

766-769.

M

A

van de Lavoir, M.C., Collarini, E.J., Leighton, P.A., Fesler, J., Lu, D.R., Harriman, W.D.,

primordial

germ

cells.

PLoS

One

7(5)

e35664.

TE

cultured

D

Thiyagasundaram, T.S., Etches, R.J., 2012. Interspecific germline transmission of

EP

doi:10.1371/journal.pone.0035664.

CC

Wernery, U., Liu, C., Baskar, V., Guerineche, Z., Khazanehdari, K.A., Saleem, S., Kinne, J., Wernery, R., Griffin, D.K. Chang, I.K., 2010. Primordial germ cell-mediated chimera

A

technology produces viable pure-line Houbara Bustard offspring: potential for repopulating

an

endangered

species.

PLoS

ONE

5(12)

e15824.

doi:10.1371/journal.pone.0015824. Zhao, D.F., Kuwana, T., 2003. Purification of avian circulating primordial germ cells by

SC

RI

PT

Nycodenz density gradient centrifugation. Brit. Poult. Sci. 44, 30-35.

Number of eggs treated

Number (%) of eggs surviving at day 2.5

Cultured PGCs

432

340 (78.7)

121 (28.0)

57

Frozen-thawed cultured PGCs

36

32 (88.9)

11 (30.6)

5

Freshly collected PGCs

72

56 (77.8)

20 (27.8)

8

Number (%) of chicks Number of matured and hatched test mated birds

CC

EP

Figure legends

TE

D

M

N

Transferred PGCs

A

U

Table 1. Survival and hatching rates of manipulated embryos and chickens

Fig. 1.

The PGC cultured in vitro. PGC isolated from embryonic blood were cultured on

A

feeder cells derived from chick embryonic fibroblast. The proliferated PGC formed colonies (A: Day 14) and most of the isolated cultured PGC (B: Day 14) maintained the typical morphology for PGC, large cells with an eccentrically positioned nucleus and a considerable amount of lipid droplets in the cytoplasm. After passages, cultured PGC proliferated and

formed colonies (C: Day 28) and most of the isolated cultured PGC (D: Day 28) also maintained the morphology of PGC. After several passages, cultured PGC continued to proliferate and formed colonies (E: Day 40, F: Day 50). Frozen-thawed cultured PGC (31 days culture, 8 days freezing and 36 days culture) proliferated and formed colonies (G) and

Proliferation of cultured PGC in vitro. PGC (about 3,000) isolated from embryonic

RI

Fig. 2.

PT

after dissociation cells had a typical PGC morphology (H). Scale bars indicate 20 µm.

SC

blood were cultured for 10 days, then sub-cultured every 5 days. Four lines were assayed in

A

Presence of female cells in cultured populations of PGC. PGC isolated from

M

Fig. 3.

N

U

separate experiments. Bars indicate standard deviations.

D

embryonic blood were cultured in vitro for 90 to 97 days. The cultured PGC were isolated

TE

from feeder cells and DNA was extracted. Presence of the W chromosome-specific repeating

EP

sequences was analysed by PCR. W-sequences were detected in cultured populations of PGC

CC

that were analysed. Lane 1: size marker, Lane 2: male (positive control), Lane 3: female (positive control), Lane 4: DW (negative control), Lanes 5-7: PGC cultured for 90, 93 and 97

A

days, Lane 8: size marker.

Fig. 4.

Colonies of cultured PGC stained with anti-CVH antibody. PGC isolated from

embryonic blood were cultured in vitro for 90 days. Cells proliferated and formed colonies.

Most of the colonies of PGC were CVH-positive (A), but some PGC colonies were CVH-negative (B). Arrows indicate PGC colonies. Scale bars indicate 20 µm.

Fig. 5.

Migration of cultured PGC to recipient gonads. PGC cultured for 31 days were

labelled with fluorescent dye (PKH26) and transferred to the Stage X blastoderm or

PT

bloodstream of recipient embryos. The manipulated embryos were cultured up to Day 7.5 of

RI

incubation in host eggshells. The fluorescent labelled PGC (red-coloured cells) successfully

SC

migrated into recipient gonads by transferring into the Stage X blastoderm (A, B) or to the

U

bloodstream (C, D). A, C: Bright light and B, D: Fluorescent light. Frozen-thawed cultured

N

PGC (31 days culture, 8 days freezing and 36 days culture) were labelled with fluorescent dye

M

A

(PKH67) and transferred to the bloodstream of recipient embryos. The manipulated embryos

D

were cultured up to Day 7.5 of incubation in host eggshells. The fluorescent labelled PGC

TE

(green-coloured cells) were observed into the gonads of recipient embryos (E: male, F:

CC

EP

female). Scale bars indicate 1 mm.

Fig. 6.

Detection of donor PGC-derived cells in recipient gonads by PCR. PGC (BPR)

A

isolated from embryonic blood were cultured on feeder cells for 90 to 97 days and transferred into the bloodstream of recipient embryos (WL). The manipulated embryos were further cultured up to Day 19.5 of incubation. The transferred donor PGC-derived cells were detected in gonads but not in livers of recipient embryos. Lane 1: size marker, Lane 2: WL (positive

control), Lane 3: BPR (positive control), Lane 4: DW (negative control), Lanes 5, 7, 9: gonads, Lanes 6, 8, 10: liver, Lanes 11, 12: gonads and liver of control embryo (no PGC transfer), Lane 13: size marker.

Fig. 7.

Detection of donor PGC-derived spermatozoa in putative germline chimaeric

PT

chickens produced by transfer of cultured PGC. The PGC isolated from embryonic blood

RI

were cultured in vitro for 40 to 90 days and then transferred to recipient embryos. The hatched

SC

chicks were raised until sexual maturity and semen was obtained from the male putative

U

germline chimaeric chickens. The sperm DNA was analysed for the presence of donor PGC

N

derived spermatozoa. Lane 1: size marker, Lane 2: WL (positive control), Lane 3: BPR

M

A

(positive control), Lane 4: DW (negative control), Lanes 5-6: germline chimaeras produced

D

by transfer of freshly collected PGC, Lanes 7-11: putative germline chimaeras, Lane 12: size

Chicks derived from cultured PGC and frozen-thawed cultured PGC. The PGC

CC

Fig. 8.

EP

TE

marker.

(BPR) cultured for 112 days were transferred to the recipient embryos (WL) and gave rise to

A

viable offspring via germline chimaeric chickens (A). Also, PGC cultured for 76 days, frozen stored in liquid nitrogen for 9 days and again cultured for 44 days after thawing were transferred to the recipient embryos and gave rise to viable offspring via germline chimaeric chickens (B). Black chicks indicate that they were derived from transferred donor PGC.

Table 2 Test mating of the germline chimaeras produced by transfer of cultured primordial germ cells from Barred Plymouth Rock to White Leghorn Number of PGCs transferred

Test period (weeks)

Number of eggs incubated

Number of chicks tested

15-112

500

17-30

91-314

49-289

49-289

0

0.0

76-9-44** 129-16-25**

500 500

16 17

81 102

58 83

58 83

0 0

0.0 0.0

F1

15

500

21

64

50

32

18

36.0

F2 F3

15 34

500 500

21 90

104 192

80 159

27 0

53 159

66.3 100.0

F4 F5

35 36

500 500

50 26

114 116

94 65

28 62

66 3

70.2 4.6

F6

41

500

24

111

96

71

25

26.0

F7 F8

46 48

500 500

18 15

94 14

71 7

36 6

35 1

49.3 14.3

F9 F10

51 85

500 500

16 11

43 23

35 14

33 11

2 3

5.7 21.4

F11 F12

85 90

500 500

13 17

3 57

2 34

1 33

1 1

50.0 2.9

Identification Days in PGC number culture

Cultured PGCs

Male M1-M27 (27 males) MF1* MF2*

Number of white Number of black chicks chicks

Black chicks (%)

91

500

12

68

51

F14 F15-F30 (16 females) FF1*

112

500

11

47

42

15-112

500

10-90

48-195

76-9-44**

500

11

24

FF2*

76-9-44**

500

28

169

FF3*

129-16-25**

500

37

174

11

21.6

3

7.1

20-162

0

0.0

19

15

4

21.1

124

124

0

0.0

162

162

0

0.0

MC1

0

200

69

MC2

0

200

49

668

522

49

473

90.6

396

297

0

297

100.0

MC3 MC4

0 0

200 200

30 22

119 101

84 93

15 13

69 80

82.1 86.0

FC1 FC2

0 0

200 200

45 41

231 181

195 173

18 19

177 154

90.8 89.0

FC3

0

FC4

0

200

48

215

173

19

154

89.0

200

43

128

114

14

100

87.7

TE

M

D

Female Freshly collected PGCs

CC

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*Frozen-thawed cultured PGCs were transferred. **Culture days before freezing - Store days in liquid nitrogen - Culture days after thawing.

A

20-162

U

N

Male Freshly collected PGCs

RI

F13

SC

40

39

A

Cultured PGCs

PT

Female