Characterisation and Germline Transmission of Cultured Avian Primordial Germ Cells

Abstract

BackgroundAvian primordial germ cells (PGCs) have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds.Principal FindingsWe confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring.ConclusionsThe establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications.

Targeted gene knockout in chickens mediated by TALENs

“…At this time, chicken PGCs can be successfully maintained in vitro without the loss of germ cell integrity (32,33), and generating transgenic chickens is feasible using transposon elements in cultured chicken PGCs (18,34). This study demonstrated the creation of specific knockout chickens using TALENs.…”

piggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens

“…S2). Only male SNUhp26 PGC-transplanted roosters were test-crossed because the reverse-sexed chimeras failed to produce donor-derived offspring and the efficiency of germ-line transmission was quiet low compared with those of the single-sex chimeras of previous reports (1,3,15). Among the donor PGC-derived progenies, the transgenic chicks could be easily identified with a fluorescent excitation lamp and the detection filters because of strong GFP expression in all transgenic chick tissues, including the beak, feather pulp, feet, and muscle ( Fig.…”

“…Choi et al (2) reported 12.5-82.6% (49.0% on average) germ-line transmission by transferring mixed PGCs, and Macdonald et al (3) estimated that the frequency of donor-derived sperm was 1-30% in the semen of recipient roosters, even though the endogenous PGCs of recipient embryos were depleted by irradiation. The frequencies in these previous reports were similar to the rates of germ-line transmission obtained from chimeras after the transfer of freshly isolated or short-term cultured PGCs (15,16).…”

“…Nevertheless, an in vitro long-term culture system for stable PGC lines remains to be established in mammals, including the mouse. Only chicken PGCs have been propagated continuously in vitro without loss of germ-cell properties (1)(2)(3). Germ-line-competent PGC lines could be versatile tools for studying germ-cell biology, including the transcriptional regulation of microRNAs (4), and for creating transgenic chickens (1).…”

Cultivation and Biological Characterization of Chicken Primordial Germ Cells

“…Chicken as an acceptance model organism (Cogburn et al 2007) have advantages of population distribution and samples can be obtained easily. PGCs have been obtained mainly from germinal crescent (Petitte et al 1990, Park andHan 2012) and embryonic blood (van de Lavoir et al 2006, Macdonald et al 2010, Miyahara et al 2014, Naito et al 2015. However, their numbers have been quite limited (Park et al 2003) and couldn't provide sufficient materials for the transgenic manipulation (Shiue et al 2009).…”