Asian Journal of Andrology

: 2015  |  Volume : 17  |  Issue : 3  |  Page : 421--426

Germline-competent stem cell in avian species and its application

Jae Yong Han1, Hyung Chul Lee1, Tae Sub Park2,  
1 Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul-151 921, Korea
2 Graduate School of International Agricultural Technology and Institute of Green Bio Science and Technology, Seoul National University, Pyeongchang-gun, Gangwon-do 232-916, Korea

Correspondence Address:
Jae Yong Han
Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul-151 921


Germ cells are the only cell type in the body that can transfer genetic information to the next generation. Germline-competent stem cells can self-renew and contribute to the germ cell lineage giving rise to pluripotent stem cells under specific conditions. Hence far, studies on germline-competent stem cells have contributed to the generation of avian model systems and the conservation of avian genetic resources. In this review, we focus on previous studies on germline-competent stem cells from avian species, mainly chicken germline-competent stem cells, which have been well established and characterized. We discuss different sources of germline-competent stem cells and recent advances for the future applications in birds.

How to cite this article:
Han JY, Lee HC, Park TS. Germline-competent stem cell in avian species and its application.Asian J Androl 2015;17:421-426

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Han JY, Lee HC, Park TS. Germline-competent stem cell in avian species and its application. Asian J Androl [serial online] 2015 [cited 2022 Aug 17 ];17:421-426
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Germline competency represents the ability of cells to give rise to a functional gamete and transmit whole genetic information to the next generation. In stem cell research, this property is very crucial to be utilized for the basic researches, as well as applied biotechnology. Germline-competent stem cell has its own characteristics of stem cell potency and germline competency. Although studies on germline-competent stem cells have been mainly focused in mammals, different sources of them from other animals should be considered for the advance in animal biotechnology.

Primordial germ cells (PGCs) have many characteristics in common with embryonic stem cells (ESCs) and differences between them at the molecular level are challenging. In mouse, PGCs are capable of self-renewal, express pluripotency-related genes such as Nanog and Oct4 as well as germ cell-related genes, [1] and are regulated by similar signaling pathways as ESCs. [2] In addition, mouse PGCs and spermatogonial stem cells (SSCs) can de-differentiate into ESC-like cells, which differentiate into three germ layers, form teratomas and undergo germline transmission [3],[4] when cultured under specific conditions. In avian species, such characteristics, including germline contribution, self-renewal, expression of specific markers and differentiation ability, are common mostly in PGCs and SSCs, and partly in blastodermal cells and ESCs, which are discussed below.

In this regard, studies on germline-competent stem cells can increase our understanding of stem cell potential and differentiation. Combined with advanced biotechnologies, including germline chimera systems, transgenesis, and genome editing, germline-competent stem cells can be a powerful tool for animal biotechnology. In this review, we introduce different types of avian germline-competent stem cells, including blastodermal cells, ESCs, PGCs and SSCs, and discuss their current research progress and future applications.

 Germline-Competent Stem Cells

Germline-competent stem cells in animal species

Studies on the establishment of pluripotent cell lines, production of germline chimera and transgenic animals have contributed to animal biotechnology for increasing human welfare as well as basic biology. Using germline-competent stem cells, valuable animals and endangered species can be reproduced and conserved. When combined with transgenic technologies, economic traits for animal products can be created. In mammals, several germline-competent stem cells that can contribute to the germline have been developed, such as ESCs, embryonic germ cells (EGCs) and SSCs.

Many studies have now reported the derivation of ESCs, EGCs or SSCs in various mammals including the pig, [5],[6],[7] cow, [8],[9],[10] sheep, [11],[12] goat, [13] and horse. [14] However, excluding studies performed in the rat and mouse (laboratory animals), the germline contribution of these cells has not been fully validated despite their ability to differentiate into three germ layers in vitro and form teratomas in vivo. [15],[16],[17] Thus, in mammals including farm animals, somatic cell nuclear transfer (SCNT) has been used mainly for animal cloning and transgenesis rather than cell-mediated methods. [18]

Recently, cell reprogramming techniques in vitro were developed to differentiate somatic cells into pluripotent stem cells, called induced pluripotent stem cells (iPSCs). [19] These cells can differentiate into the three germ layers as well as into germ cells in vitro and in vivo, similarly to ESCs. Initially, iPSCs were generated from embryonic and adult somatic cells by introduction of several pluripotency-related genes such as Oct4, Sox2, cMyc and Klf4. [19],[20] Since iPSCs can be generated from adult cells, they are thought to be an alternative tool to ESCs for basic stem cell studies and human disease models. For animals in which pluripotent cell lines have not been established, iPSCs are a good alternative to animal transgenesis. In addition to mice and humans, many studies on the generation of iPSCs from mammals, including the monkey [21] and pig, [22] have been reported. Since iPSCs can be induced to differentiate into germ cells in vitro, [23],[24] the establishment of iPSCs from domestic animals can be useful for animal transgenesis using germline chimera techniques. In nonmammalian species, derivation of avian iPSCs has been recently reported. [25],[26] In particular, induced pluripotency in chicken embryonic fibroblasts resulted in a germ cell fate, suggesting different cellular mechanisms between aves and mammals. [25]

Germline-competent stem cells in avian species

Importance of germline-competent stem cell study in avian species

The most common and powerful tool for transgenesis in mammals is pronuclear injection and SCNT techniques using unfertilized oocytes. In avian species, however, several obstacles due to the unique characteristics of avian eggs make it difficult to apply conventional systems. The cytoplasm of avian oocytes is very large compared with other species and is connected to yolk, which results in imperfect cellularization. [27] The yolky and large cytoplasm of avian oocyte makes it difficult to perform pronuclear injection of exogenous DNA. Due to the large amount of yolk, avian eggs have no transparency and it is difficult to identify the nucleus of oocytes. Thus, SCNT has not been possible in avians. Therefore, germline-competent stem cells-mediated transgenic systems are required. [28] Due to the unique developmental features of avian species, it is easy to observe and manipulate avian embryos compared with other species. Thus, germ cells and embryos at any stage can be monitored and isolated easily. It has been demonstrated experimentally that several germline-competent stem cells derived from different developmental stages can contribute to the germline and have self-renewal activity in chickens. At this time, several germline-competent stem cells such as blastodermal cells, ESCs, PGCs and SSCs have been used for germline chimera production and transgenesis in avian species ([Table 1]). Therefore, for practical applications, it is important to understand the origin of each cell type and the related techniques.{Table 1}

Germ cell development in avian species

In avian species, different types of germline-competent stem cells exist and can be obtained according to their developmental stages. Thus, understanding the developmental process of germ cells is required. In chickens, although PGC specification remains unclear at this time, germ plasm structures present in oocytes and cleavage stage embryos via expression of chicken vasa homolog (CVH), which is strong evidence of the preformation mode, have been reported. [29],[30] During primitive streak formation, PGCs migrate gradually toward the anterior region and the germinal crescent. [31] They incorporate into blood vessels at Hamburger, and Hamilton (HH) stages 10-12 [32],[33] and undergo circulation at HH stages 12-15 before settling into embryonic gonads. [34],[35] The chicken PGCs then migrate toward the gonadal ridges at HH stage 17 to differentiate. [34] In the adult testis, spermatogonia including SSCs are located close to the basal membrane of the seminiferous tubules.

Blastodermal cells and embryonic stem cells

In chickens, after fertilization in the isthmus and following vigorous cell division for 20 h in the uterus, blastoderms containing approximately 40 000-60 000 cells develop at Eyal-Giladi and Kochav (EGK) stage X, which is the oviposition stage. [36] At EGK stage X, 1-2 cell layers are present in the area pellucida (a central region), while the area opaca (a peripheral region) remains multilayered. Although the anteroposterior embryonic axis is already formed at EGK stage X, the blastodermal cells are undifferentiated. When the cells were transplanted into the subgerminal cavity of recipient embryos at EGK stage X, somatic and germline chimeras were produced successfully, demonstrating their stem cell characteristics. [37],[38],[39]

Another utilization of blastodermal cells is for in vitro culture to generate pluripotent cell lines such as ESCs, which are derived from the inner cell mass of the blastocyst in mammals. [40],[41] Blastodermal cells were cultured in medium containing leukemia inhibitory factor (LIF), stem cell factor, insulin growth factor 1 (IGF-1) and other factors, [42],[43] which is conventional culture medium used for murine ESCs, to form chicken ESCs. Chicken ESCs express several stem cell markers such as stage-specific embryonic antigen-1 (SSEA-1), alkaline phosphatase and epithelial membrane antigen-1 (EMA-1). [42],[44],[45] In addition, chicken ESCs can differentiate into three germ layers and form embryoid bodies through the removal of LIF from the culture medium, and produce somatic chimeras when they are reintroduced into recipient embryos, indicative of their pluripotency. [42],[45]

Primordial germ cells

Primordial germ cells are precursors of functional gametes, sperm or eggs. PGCs in most animal species originate from early developmental stages through two different mechanisms; the preformation and induction modes. [29] In chickens, PGCs have been thought to originate through the preformation mode by maternally inherited determinants such as CVH. [30] At HH stage 4, when the primitive streak is fully developed, PGCs gather in the germinal crescent, which is the anterior marginal region between the area opaca and area pellucida. [35] The PGCs then circulate in the extraembryonic blood vessels until they settle in the genital ridges at HH stage 17. [34] After embryonic day 6, when gonadal sex differentiation occurs, the PGCs undergo sex-specific differentiation. [46] In this regard, until embryonic day 6, PGCs can be isolated from different embryonic tissues such as the germinal crescent at HH stage 4, extraembryonic blood vessels at embryonic day 2.2 and embryonic gonads at embryonic day 5-6. To enrich of the isolated PGCs, fluorescence-activated cell sorting and magnetic-activated cell sorting using PGC-specific antibodies have been used in avian systems. [47],[48]

Since only a limited number of PGCs can be obtained from embryos, it is important to establish long-term in vitro culture systems for PGCs. Recent studies demonstrated the establishment of such systems for chicken PGCs using germline transmission efficiency. [49],[50],[51] It is demonstrated that basic fibroblast growth factor (bFGF) via MEK/ERK signaling plays a crucial role in the survival and long-term proliferation of chicken PGCs. [49] In addition, cultured PGCs express PGC-specific markers and exhibit telomerase activity, migratory activity, and germline contribution, indicating that germ cell integrity can be maintained even after long-term culture. [49],[51] In this regard, in avian species, PGCs are the most powerful type of germline-competent stem cell for avian biotechnology.

Spermatogonial stem cells

After entering embryonic gonads, PGCs undergo sex-specific differentiation: spermatogenesis in males and oogenesis in females. In male chickens, PGCs enter mitotic arrest as prospermatgonia at embryonic day 8, and spermatogonia proliferate after hatching. [46] SSCs, self-renewing cells in the testis that can produce sperm continuously, are classified into four types in avians based on their morphological characteristics: a dark type A (Ad), two pale A types (Ap1 and Ap2) and dark type B spermatogonia. [52] Among these, the Ad spermatogonia are thought to be SSCs.

In chicken, SSCs can be isolated from juveniles at 3-4 weeks or from adult testes. The proportions of SSCs are approximately 0.4% in juvenile testis and 0.03% in adult testis. [53] When chicken testicular cells were cultured in medium containing LIF, bFGF and IGF-1, they formed colony structures and expressed germ cell-specific markers such as SSEA-1/3/4, integrin alpha 6, integrin beta 1 and EMA-1. In addition, these SSCs differentiated into embryoid bodies in LIF-free medium, indicative of their stem cell potency. [53]

 Application of Germline-Competent Stem Cells

Germline chimera and transgenic birds

Blastodermal cells and embryonic stem cells

In many studies on germline chimera and transgenic birds, researchers have targeted blastodermal cells or blastoderms at EGK stage X. Compared with PGCs or SSCs, blastodermal cells can easily be obtained and manipulated from fertilized eggs. When retroviral or lentiviral vectors were introduced into EGK stage X embryos, transgene expression was observed in various tissues (including germ cells) after further development, indicative of the contribution of blastodermal cells to somatic and germ cells. [54],[55] In addition, when isolated blastodermal cells were transplanted into the subgerminal cavity of EGK stage X embryos, somatic and germline chimeras were produced. [37],[39] However, compared with the somatic chimerism rate, germline transmission efficiency of blastodermal cells was too low.

In another approach, when chicken ESCs were transplanted after in vitro transfection, transgene-expressing chimeric chickens were produced. [56] Despite several efforts with in vitro culture of chicken ESCs, germline transmission capacity was extremely low and has not been repeated experimentally so far. [57] One possible reason is that chicken germ cell and somatic lineages are already segregated at EGK stage X, according to the presence of the CVH protein and germplasm-like structures in chicken oocytes and cleavage stage embryos, [30] such that an insufficient number of PGCs among blastodermal cells exists to contribute to the germline. In addition, chicken ESCs showed a reduction in germline potency during culture, but then reacquired potency when exogenous CVH was overexpressed, [58] indicating the conventional culture condition used for ESCs cannot induce germ cell fate. Thus, to utilize blastodermal cells or ESCs from the chicken, greater detail on germ cell formation is required.

Primordial germ cells

In avian species, PGCs are more commonly used to produce the germline chimera than ESCs, SSCs and other germline-competent cells because of their germline unipotency and easy access. Especially, germline chimeras in chicken and quail have been produced using blood PGCs and gonadal PGCs after transplantation into the recipient embryonic blood vessels. [48],[51],[59],[60],[61],[62] Since other pluripotent stem cell lines such as ESCs and EGCs in chicken showed too low germline competency, PGCs are thought to be an optimal tool for a stable avian transgenesis. [42],[63],[64]

The establishment of long-term culture techniques for PGCs has resulted in remarkable advances in avian biotechnology. Using nonviral transfection of cultured PGCs, transgenes were expressed continuously and strongly in the transgenic chickens for generations. [65],[66],[67] More recently, gene knockout chickens were produced by genome editing on PGCs using transcription activator-like effector nucleases (TALENs) and homologous recombination. [68],[69] In particular, because TALEN knockout chickens are nontransgenic, they can be widely used for agricultural practical applications. [68] Thus, the next goal in avian biotechnology will be the development of elaborate and efficient genome editing techniques for model animals. [68] One possible method is the Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR)-Cas system. [70],[71] Because the CRISPR-Cas system is more efficient for the insertion, deletion or modification of target genes compared with TALENs, it requires further investigation before application in avian systems.

Spermatogonial stem cells

To produce transgenic birds using blastodermal cells, ESCs or PGCs, it is important to wait until the founder's sexual maturation and to produce a large number of founders for enrichment and analysis of donor-derived offspring. Thus, although studies on avian SSCs are limited compared with blastodermal cells and PGCs, utilization of SSCs can be a time-efficient alternative. When testicular cells were transplanted into recipient testes, they could produce donor-derived offspring. [72],[73] In addition, when freshly isolated or in vitro cultured SSCs were transplanted into blastoderms at EGK stage X or embryonic blood vessels at 53-54 h, they could produce donor-derived offspring, indicating that later-stage germ cells maintain their developmental unipotency in vivo. [74] One major obstacle of SSCs for avian biotechnology is their low germline transmission efficiency. Despite this, the transgenic efficiency of offspring derived from transplanted SSCs is high. Thus, studies on the enrichment of exogenous SSCs in recipient testes and maintenance of their potency during culture are required. On the other hand, another candidate can be oogonial stem cells (OSCs) which are present in adult ovary. After the first studies on mammalian OSCs were reported at 2004, [75],[76],[77] isolation and characterization of them has been actively studied. However, there is no report on OSCs in avian species, so far. Therefore, investigation of reliable markers of avian OSCs should be studied in the future.

Interspecies germline chimera for restoration of endangered species

Endangered species, which increase every year, are an important and global problem that must be addressed. Although animal cloning using nuclear transfer has been developed in mammals, it is not applicable to avian species. Thus, an interspecies germline chimera system is a powerful tool for the conservation of avian genetic resources. At this time, many studies have been conducted on the production of interspecies germline chimeras by transplantation of PGCs, blastodermal cells and SSCs, including quail-to-chicken, [78],[79],[80] turkey-to-chicken, [81] duck-to-chicken [82] chicken-to-duck [83] and chicken-to-guinea fowl. [84] However, the production of donor-derived progeny was too low [82] or absent, [78],[79],[81] mainly due to physiological differences between species such as reproduction cycle and egg size. Recently, donor-derived progeny of wild birds, including pheasant [85] and Houbara bustard [86] using PGCs and gonadal cells, respectively, were produced successfully using the interspecies germline chimera system. Thus, for applications in other endangered birds, culture methods of germline-competent stem cells for diverse species should be investigated. In addition, physiological aspects such as donor cell sex and differences between species should be considered. [87]


Recent advances in long-term in vitro culture of germline-competent stem cells and germline chimera production in the chicken combined with genome editing have highlighted the need for an avian species as an animal model [68],[69] ([Figure 1]). In addition, the production of interspecies germline chimera via germline-competent stem cells has become a powerful tool for the restoration of endangered avian species [83],[85],[87] ([Figure 1]). Since several types of germline-competent stem cells can be obtained from different developmental stages in birds, optimal in vitro culture systems for different types of germline-competent stem cells should be established in future studies. Indeed, the presence and function of germline-competent stem cells are closely associated with the potency of the cells. In other words, once they emerge, understanding why and how they acquire their potency is important to regulate and maintain these cells in vitro. Therefore, studies on the underlying mechanisms of germline-competent stem cells formation and maintenance in avian species can contribute to the utilization of avian species as animal model systems and the conservation of avian genetic resources.{Figure 1}

 Author Contributions

JYH conceived of the study, and JYH, HCL and TSP participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

 Competing Interests

All authors declare no competing interests.


This work was supported by a grant from the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ010390012014 and Project No. PJ01053301) of the Rural Development Administration, Republic of Korea.


1Mise N, Fuchikami T, Sugimoto M, Kobayakawa S, Ike F, et al. Differences and similarities in the developmental status of embryo-derived stem cells and primordial germ cells revealed by global expression profiling. Genes Cells 2008; 13: 863-77.
2De Felici M, Farini D, Dolci S. In or out stemness: comparing growth factor signalling in mouse embryonic stem cells and primordial germ cells. Curr Stem Cell Res Ther 2009; 4: 87-97.
3Guan K, Nayernia K, Maier LS, Wagner S, Dressel R, et al. Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 2006; 440: 1199-203.
4Labosky PA, Barlow DP, Hogan BL. Embryonic germ cell lines and their derivation from mouse primordial germ cells. Ciba Found Symp 1994; 182: 157-68.
5Piedrahita JA, Anderson GB, Martin GR, Bondurant RH, Pashen RL. Isolation of embryonic stem cell-like colonies from porcine embryos. Theriogenology 1988; 29: 286.
6Shim H, Gutiérrez-Adán A, Chen LR, BonDurant RH, Behboodi E, et al. Isolation of pluripotent stem cells from cultured porcine primordial germ cells. Biol Reprod 1997; 57: 1089-95.
7Marret C, Durand P. Culture of porcine spermatogonia: effects of purification of the germ cells, extracellular matrix and fetal calf serum on their survival and multiplication. Reprod Nutr Dev 2000; 40: 305-19.
8Saito S, Strelchenko N, Niemann H. Bovine embryonic stem cell-like cell-lines cultured over several passages. Roux Arch Dev Biol 1992; 201: 134-41.
9Strelchenko N. Bovine pluripotent stem cells. Theriogenology 1996; 45: 131-40.
10Oatley JM, de Avila DM, Reeves JJ, McLean DJ. Testis tissue explant culture supports survival and proliferation of bovine spermatogonial stem cells. Biol Reprod 2004; 70: 625-31.
11Handyside A, Hooper ML, Kaufman MH, Wilmut I. Towards the isolation of embryonal stem-cell lines from the sheep. Roux Arch Dev Biol 1987; 196: 185-90.
12Ledda S, Bogliolo L, Bebbere D, Ariu F, Pirino S. Characterization, isolation and culture of primordial germ cells in domestic animals: recent progress and insights from the ovine species. Theriogenology 2010; 74: 534-43.
13Hua J, Zhu H, Pan S, Liu C, Sun J, et al. Pluripotent male germline stem cells from goat fetal testis and their survival in mouse testis. Cell Reprogram 2011; 13: 133-44.
14Saito S, Ugai H, Sawai K, Yamamoto Y, Minamihashi A, et al. Isolation of embryonic stem-like cells from equine blastocysts and their differentiation in vitro. FEBS Lett 2002; 531: 389-96.
15Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292: 154-6.
16Buehr M, Meek S, Blair K, Yang J, Ure J, et al. Capture of authentic embryonic stem cells from rat blastocysts. Cell 2008; 135: 1287-98.
17Li P, Tong C, Mehrian-Shai R, Jia L, Wu N, et al. Germline competent embryonic stem cells derived from rat blastocysts. Cell 2008; 135: 1299-310.
18Niemann H, Lucas-Hahn A. Somatic cell nuclear transfer cloning: practical applications and current legislation. Reprod Domest Anim 2012; 47 Suppl 5: 2-10.
19Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126: 663-76.
20Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131: 861-72.
21Zhong B, Trobridge GD, Zhang X, Watts KL, Ramakrishnan A, et al. Efficient generation of nonhuman primate induced pluripotent stem cells. Stem Cells Dev 2011; 20: 795-807.
22Liu K, Ji G, Mao J, Liu M, Wang L, et al. Generation of porcine-induced pluripotent stem cells by using OCT4 and KLF4 porcine factors. Cell Reprogram 2012; 14: 505-13.
23Imamura M, Aoi T, Tokumasu A, Mise N, Abe K, et al. Induction of primordial germ cells from mouse induced pluripotent stem cells derived from adult hepatocytes. Mol Reprod Dev 2010; 77: 802-11.
24Zhu Y, Hu HL, Li P, Yang S, Zhang W, et al. Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study. Asian J Androl 2012; 14: 574-9.
25Lu Y, West FD, Jordan BJ, Jordan ET, West RC, et al. Induced pluripotency in chicken embryonic fibroblast results in a germ cell fate. Stem Cells Dev 2014; 23: 1755-64.
26Lu Y, West FD, Jordan BJ, Mumaw JL, Jordan ET, et al. Avian-induced pluripotent stem cells derived using human reprogramming factors. Stem Cells Dev 2012; 21: 394-403.
27Lee HC, Choi HJ, Park TS, Lee SI, Kim YM, et al. Cleavage events and sperm dynamics in chick intrauterine embryos. PLoS One 2013; 8: e80631.
28Han JY. Germ cells and transgenesis in chickens. Comp Immunol Microbiol Infect Dis 2009; 32: 61-80.
29Extavour CG, Akam M. Mechanisms of germ cell specification across the metazoans: epigenesis and preformation. Development 2003; 130: 5869-84.
30Tsunekawa N, Naito M, Sakai Y, Nishida T, Noce T. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development 2000; 127: 2741-50.
31Ginsburg M, Eyal-Giladi H. Temporal and spatial aspects of the gradual migration of primordial germ cells from the epiblast into the germinal crescent in the avian embryo. J Embryol Exp Morphol 1986; 95: 53-71.
32Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol 1951; 88: 49-92.
33Ukeshima 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) 1991; 40: 124-8.
34Fujimoto T, Ukeshima A, Kiyofuji R. The origin, migration and morphology of the primordial germ cells in the chick embryo. Anat Rec 1976; 185: 139-45.
35Swift CH. Origin and early history of the primordial germ-cells in the chick. Am J Anat 1914; 15: 483-516.
36Eyal-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 1976; 49: 321-37.
37Carsience RS, Clark ME, Verrinder Gibbins AM, Etches RJ. Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos. Development 1993; 117: 669-75.
38Marzullo G. Production of chick chimaeras. Nature 1970; 225: 72-3.
39Petitte JN, Clark ME, Liu G, Verrinder Gibbins AM, Etches RJ. Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells. Development 1990; 108: 185-9.
40Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292: 154-6.
41Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: 1145-7.
42Pain B, Clark ME, Shen M, Nakazawa H, Sakurai M, et al. Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities. Development 1996; 122: 2339-48.
43Petitte JN, Liu G, Yang Z. Avian pluripotent stem cells. Mech Dev 2004; 121: 1159-68.
44Park HJ, Park TS, Kim TM, Kim JN, Shin SS, et al. Establishment of an in vitro culture system for chicken preblastodermal cells. Mol Reprod Dev 2006; 73: 452-61.
45van de Lavoir MC, Mather-Love C, Leighton P, Diamond JH, Heyer BS, et al. High-grade transgenic somatic chimeras from chicken embryonic stem cells. Mech Dev 2006; 123: 31-41.
46Zheng YH, Rengaraj D, Choi JW, Park KJ, Lee SI, et al. Expression pattern of meiosis associated SYCP family members during germline development in chickens. Reproduction 2009; 138: 483-92.
47Ono T, Machida Y. Immunomagnetic purification of viable primordial germ cells of Japanese quail (Coturnix japonica). Comp Biochem Physiol A Mol Integr Physiol 1999; 122: 255-9.
48Park TS, Jeong DK, Kim JN, Song GH, Hong YH, et al. Improved germline transmission in chicken chimeras produced by transplantation of gonadal primordial germ cells into recipient embryos. Biol Reprod 2003; 68: 1657-62.
49Choi JW, Kim S, Kim TM, Kim YM, Seo HW, et al. Basic fibroblast growth factor activates MEK/ERK cell signaling pathway and stimulates the proliferation of chicken primordial germ cells. PLoS One 2010; 5: e12968.
50Macdonald J, Glover JD, Taylor L, Sang HM, McGrew MJ. Characterisation and germline transmission of cultured avian primordial germ cells. PLoS One 2010; 5: e15518.
51van de Lavoir MC, Diamond JH, Leighton PA, Mather-Love C, Heyer BS, et al. Germline transmission of genetically modified primordial germ cells. Nature 2006; 441: 766-9.
52Lin M, Jones RC. Renewal and proliferation of spermatogonia during spermatogenesis in the Japanese quail, Coturnix coturnix japonica. Cell Tissue Res 1992; 267: 591-601.
53Jung JG, Lee YM, Park TS, Park SH, Lim JM, et al. Identification, culture, and characterization of germline stem cell-like cells in chicken testes. Biol Reprod 2007; 76: 173-82.
54Bosselman RA, Hsu RY, Boggs T, Hu S, Bruszewski J, et al. Germline transmission of exogenous genes in the chicken. Science 1989; 243: 533-5.
55McGrew MJ, Sherman A, Ellard FM, Lillico SG, Gilhooley HJ, et al. Efficient production of germline transgenic chickens using lentiviral vectors. EMBO Rep 2004; 5: 728-33.
56Zhu L, van de Lavoir MC, Albanese J, Beenhouwer DO, Cardarelli PM, et al. Production of human monoclonal antibody in eggs of chimeric chickens. Nat Biotechnol 2005; 23: 1159-69.
57Lavial F, Pain B. Chicken embryonic stem cells as a non-mammalian embryonic stem cell model. Dev Growth Differ 2010; 52: 101-14.
58Lavial F, Acloque H, Bachelard E, Nieto MA, Samarut J, et al. Ectopic expression of Cvh (chicken vasa homologue) mediates the reprogramming of chicken embryonic stem cells to a germ cell fate. Dev Biol 2009; 330: 73-82.
59Kim JN, Park TS, Park SH, Park KJ, Kim TM, et al. Migration and proliferation of intact and genetically modified primordial germ cells and the generation of a transgenic chicken. Biol Reprod 2010; 82: 257-62.
60Kim MA, Park TS, Kim JN, Park HJ, Lee YM, et al. Production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells into recipient embryos. Theriogenology 2005; 63: 774-82.
61Ono T, Matsumoto T, Arisawa Y. Production of donor-derived offspring by transfer of primordial germ cells in Japanese quail. Exp Anim 1998; 47: 215-9.
62Tajima A, Naito M, Yasuda Y, Kuwana T. Production of germ line chimera by transfer of primordial germ cells in the domestic chicken (Gallus domesticus). Theriogenology 1993; 40: 509-19.
63Park TS, Han JY. Derivation and characterization of pluripotent embryonic germ cells in chicken. Mol Reprod Dev 2000; 56: 475-82.
64Park TS, Hong YH, Kwon SC, Lim JM, Han JY. Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos. Mol Reprod Dev 2003; 65: 389-95.
65Park TS, Han JY. PiggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens. Proc Natl Acad Sci U S A 2012; 109: 9337-41.
66Macdonald J, Taylor L, Sherman A, Kawakami K, Takahashi Y, et al. Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons. Proc Natl Acad Sci U S A 2012; 109: e1466-72.
67Leighton PA, van de Lavoir MC, Diamond JH, Xia C, Etches RJ. Genetic modification of primordial germ cells by gene trapping, gene targeting, and phiC31 integrase. Mol Reprod Dev 2008; 75: 1163-75.
68Park TS, Lee HJ, Kim KH, Kim JS, Han JY. Targeted gene knockout in chickens mediated by TALENs. Proc Natl Acad Sci U S A 2014; 111: 12716-21.
69Schusser B, Collarini EJ, Yi H, Izquierdo SM, Fesler J, et al. Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells. Proc Natl Acad Sci U S A 2013; 110: 20170-5.
70Cong L, Ran FA, Cox D, Lin S, Barretto R, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339: 819-23.
71Cho SW, Kim S, Kim JM, Kim JS. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 2013; 31: 230-2.
72Lee YM, Jung JG, Kim JN, Park TS, Kim TM, et al. A testis-mediated germline chimera production based on transfer of chicken testicular cells directly into heterologous testes. Biol Reprod 2006; 75: 380-6.
73Trefil P, Micáková A, Mucksová J, Hejnar J, Poplstein M, et al. Restoration of spermatogenesis and male fertility by transplantation of dispersed testicular cells in the chicken. Biol Reprod 2006; 75: 575-81.
74Jung JG, Lee YM, Kim JN, Kim TM, Shin JH, et al. The reversible developmental unipotency of germ cells in chicken. Reproduction 2010; 139: 113-9.
75Johnson J, Canning J, Kaneko T, Pru JK, Tilly JL. Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 2004; 428: 145-50.
76Zou K, Yuan Z, Yang Z, Luo H, Sun K, et al. Production of offspring from a germline stem cell line derived from neonatal ovaries. Nat Cell Biol 2009; 11: 631-6.
77Tilly JL, Telfer EE. Purification of germline stem cells from adult mammalian ovaries: a step closer towards control of the female biological clock? Mol Hum Reprod 2009; 15: 393-8.
78Ono T, Yokoi R, Aoyama H. Transfer of male or female primordial germ cells of quail into chick embryonic gonads. Exp Anim 1996; 45: 347-52.
79Watanabe M, Kinutani M, Naito M, Ochi O, Takashima Y. Distribution analysis of transferred donor cells in avian blastodermal chimeras. Development 1992; 114: 331-8.
80Roe M, McDonald N, Durrant B, Jensen T. Xenogeneic transfer of adult quail (Coturnix coturnix) spermatogonial stem cells to embryonic chicken (Gallus gallus) hosts: a model for avian conservation. Biol Reprod 2013; 88: 129.
81Reynaud G. The transfer of turkey primordial germ cells to chick embryos by intravascular injection. J Embryol Exp Morphol 1969; 21: 485-507.
82Li ZD, Deng H, Liu CH, Song YH, Sha J, et al. Production of duck-chicken chimeras by transferring early blastodermal cells. Poult Sci 2002; 81: 1360-4.
83Liu C, Khazanehdari KA, Baskar V, Saleem S, Kinne J, et al. Production of chicken progeny (Gallus gallus domesticus) from interspecies germline chimeric duck (Anas domesticus) by primordial germ cell transfer. Biol Reprod 2012; 86: 101.
84van de Lavoir MC, Collarini EJ, Leighton PA, Fesler J, Lu DR, et al. Interspecific germline transmission of cultured primordial germ cells. PLoS One 2012; 7: e35664.
85Kang SJ, Choi JW, Kim SY, Park KJ, Kim TM, et al. Reproduction of wild birds via interspecies germ cell transplantation. Biol Reprod 2008; 79: 931-7.
86Wernery U, Liu C, Baskar V, Guerineche Z, Khazanehdari KA, et al. Primordial germ cell-mediated chimera technology produces viable pure-line Houbara bustard offspring: potential for repopulating an endangered species. PLoS One 2010; 5: e15824.
87Kang SJ, Choi JW, Park KJ, Lee YM, Kim TM, et al. Development of a pheasant interspecies primordial germ cell transfer to chicken embryo: effect of donor cell sex on chimeric semen production. Theriogenology 2009; 72: 519-27.