In this work we use TALE nucleases (TALENs) to target a reporter construct to the DDX4 (vasa) locus in chicken primordial germ cells (PGCs). Vasa is a key germ cell determinant in many animal species and is posited to control avian germ cell formation. We show that TALENs mediate homology-directed repair of the DDX4 locus on the Z sex chromosome at high (8.1%) efficiencies. Large genetic deletions of 30 kb encompassing the entire DDX4 locus were also created using a single TALEN pair. The targeted PGCs were germline competent and were used to produce DDX4 null offspring. In DDX4 knockout chickens, PGCs are initially formed but are lost during meiosis in the developing ovary, leading to adult female sterility. TALEN-mediated gene targeting in avian PGCs is therefore an efficient process.

/pdf/efficient-talen-mediated-gene-targeting-of-chicken-2rt9w7mc6b.pdf

Efficient TALEN-mediated gene targeting of chicken primordial germ cells.

Germ cells are the only cell type capable of transmitting genetic information to the next generation. During development, they are set aside from all somatic cells of the embryo. In many species, germ cells form at the fringe of the embryo proper and then traverse through several developing somatic tissues on their migration to the emerging gonads. Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. Unlike other species, in avian embryos, PGCs use blood circulation for transport to the future gonadal region. This unique accessibility of avian PGCs during early development provides an opportunity to collect and transplant PGCs. The recent development of methods for production of germline chimeras by transfer of PGCs, and long-term cultivation methods of chicken PGCs without losing their germline transmission ability have provided important breakthroughs for the preservation of germplasm , for the production of transgenic birds and study the germ cell system. This review will describe the development, migration, differentiation and manipulation of germ cells, and discuss the prospects that germ cell technologies offer for agriculture, biotechnology and academic research.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/dgd.12026

Development, differentiation and manipulation of chicken germ cells

Chronic fatigue syndrome (CFS) is a multisystem disorder characterized by prolonged and severe fatigue that is not relieved by rest. Attempts to treat CFS have been largely ineffective primarily because the etiology of the disorder is unknown. Recently, CFS has been associated with xenotropic murine leukemia virus-related virus (XMRV) as well as other murine leukemia virus (MLV)-related viruses, though not all studies have found these associations. We collected blood samples from 100 CFS patients and 200 self-reported healthy volunteers from the same geographical area. We analyzed these in a blind manner using molecular, serological, and viral replication assays. We also analyzed samples from patients in the original study that reported XMRV in CFS patients. We did not find XMRV or related MLVs either as viral sequences or infectious viruses, nor did we find antibodies to these viruses in any of the patient samples, including those from the original study. We show that at least some of the discrepancy with previous studies is due to the presence of trace amounts of mouse DNA in the Taq polymerase enzymes used in these previous studies. Our findings do not support an association between CFS and MLV-related viruses, including XMRV, and the off-label use of antiretrovirals for the treatment of CFS does not seem justified at present.

https://journals.asm.org/doi/pdf/10.1128/JVI.00693-11

Absence of XMRV retrovirus and other murine leukemia virus-related viruses in patients with chronic fatigue syndrome

Background The Houbara bustard (Chlamydotis undulata) is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Afric population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, h reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpo investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring. Methodology/Principal Findings Embryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue germ cells (gPGCs) was injected into White Leghorn chicken (Gallus gallus domesticus) embryos, producing 83/138 surviving male ch chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken with Houbara-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor H semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which wer male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Hou rooster.

/pdf/primordial-germ-cell-mediated-chimera-technology-produces-80o3pgrldo.pdf

Primordial Germ Cell-Mediated Chimera Technology Produces Viable Pure-Line Houbara Bustard Offspring: Potential for Repopulating an Endangered Species

To investigate germline development and germ cell specification, we identified a Dazl homolog (CagDazl) from gynogenetic gibel carp (Carassius auratus gibelio) Its cDNA sequence and BAC clone sequence analyses revealed the genomic organization conservation and conserved synteny of the Dazl family members and their neighborhood genes among vertebrates, especially in fish Moreover, a polyclonal antibody specific to CagDazl was produced and used to examine its expression and distribution throughout germline development at protein level Firstly, ovary-specific expression pattern of CagDazl was confirmed in adult tissues by RT-PCR and Western blot In addition, in situ hybridization and immunofluorescence localization demonstrated its specific expression in germ cells, and both its transcript and protein were localized to germ plasm Then, co-localization of CagDazl and mitochondrial cloud was found, confirming that CagDazl transcript and its protein are germ plasm component and move via METRO pathway during oogenesis Furthermore, the CagDazl is abundant and continuous throughout germline development and germ cell specification including primordial germ cell (PGC) formation, oogonium differentiation, oocyte development, and embryogenesis, and the dynamic distribution occurs at different development stages The data suggest that maternal CagDazl might play an important role in gibel carp PGC formation Therefore, CagDazl is a useful and specific marker for tracing germ plasm and germ cell development in the gynogenetic gibel carp In addition, in comparison with previous studies in sexual reproduction species, the continuous and dynamic distribution of CagDazl protein in the germ plasm throughout the life cycle seems to have significant implication in sex evolution of vertebrates

Evolutionary conservation of Dazl genomic organization and its continuous and dynamic distribution throughout germline development in gynogenetic gibel carp.

A novel method was developed to isolate chick primordial germ cells (PGCs) from circulating embryonic blood. This is a very simple and rapid method for the isolation of circulating PGCs (cPGCs) using an ammonium chloride-potassium (ACK) buffer for lysis of the red blood cells. The PGCs were purified as in vitro culture proceeded. Most of the initial red blood cells were removed in the first step using the ACK lysis buffer. The purity of the cPGCs after ACK treatment was 57.1%, and the recovery rate of cPGCs from whole blood was 90.3%. The ACK process removed only red blood cells and it did not affect cPGC morphology. In the second step, the red blood cells disappeared as the culture progressed. At 7 days of in vitro culture, the purity of the PGCs was 92.9%. Most of these cells expressed germline-specific antibodies, such as those against chicken vasa homolog (CVH). The cultured PGCs expressed the Cvh and Dazl genes. Chimeric chickens were produced from these cultured PGCs, and the donor cells were detected in the gonads, suggesting that the PGCs had biological function. In conclusion, this novel isolation system for PGCs should be easier to use than previous methods. The results of the present study suggest that this novel method will become a powerful tool for germline manipulation in the chicken.

https://bioone.org/journals/Biology-of-Reproduction/volume-77/issue-1/biolreprod.107.061200/A-Novel-Method-to-Isolate-Primordial-Germ-Cells-and-Its/10.1095/biolreprod.107.061200.pdf

A Novel Method to Isolate Primordial Germ Cells and Its Use for the Generation of Germline Chimeras in Chicken

The aim of the present study was to improve the efficiency of endogenous primordial germ cell (PGC) depletion and to increase the ratio of donor PGCs in the gonads of recipient chicken embryos. A sustained-release emulsion was prepared by emulsifying equal amounts of Ca2+- and Mg2+-free phosphate-buffered saline containing 10% busulfan solubilised in N,N-dimethylformamide and sesame oil, using a filter. Then, 75 μg per 50 μL busulfan sustained-release emulsion was injected into the yolk. To determine the depletion and repopulation of PGCs in the gonads after 6 days incubation, whole-mount immunostaining was performed. The busulfan sustained-release emulsion significantly reduced the number of endogenous PGCs compared with control (P < 0.05). Moreover, the busulfan sustained-release emulsion significantly depleted endogenous PGCs compared with other previously reported busulfan delivery systems (P < 0.05), but with less variation, suggesting that the sustained-release emulsion delivered a consistent amount of busulfan to the developing chicken embryos. The PGC transfer study showed that the proportion of donor PGCs in the gonads of busulfan sustained-release emulsion-treated embryos after 6 days incubation increased 28-fold compared with control. In conclusion, the results demonstrate that exogenous PGCs are capable of migrating and settling in gonads from which endogenous PGCs have been removed using a busulfan sustained-release emulsion.

Increased proportion of donor primordial germ cells in chimeric gonads by sterilisation of recipient embryos using busulfan sustained-release emulsion in chickens

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A Novel Method to Isolate Primordial Germ Cells and Its Use for the Generation of Germline Chimeras in Chicken

Increased proportion of donor primordial germ cells in chimeric gonads by sterilisation of recipient embryos using busulfan sustained-release emulsion in chickens