Transplantation Of Primordial Germ Cells Research Articles

Retraction Note: Production of viable chicken by allogeneic transplantation of primordial germ cells induced from somatic cells

Retraction Note: Production of viable chicken by allogeneic transplantation of primordial germ cells induced from somatic cells

Transplantation and enrichment of busulfan-resistant primordial germ cells into adult testes for efficient production of germline chimeras in songbirds†.

Zebra finch is a unique model for behavioral, neural, and genomic studies of vocal learning. Several transgenic zebra finches have been produced, although the germline transmission efficiencies are reportedly low. Recently, there have been attempts to produce germline chimeras using primordial germ cells (PGCs). However, this has been hampered by difficulties associated with the manipulation of the small eggs and the fact that the zebra finch is an altricial species that requires parental care after birth, unlike precocial chickens. Consequently, it is difficult to transplant PGCs into embryos and maintain the chimeras. Here, we developed a busulfan-mediated system for transplantation of PGCs into adult testes, to produce germline chimeras with an improved germline transmission capacity. We established microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to busulfan, which induces germ cell-specific cytotoxicity, and transplanted them into testes rendered temporarily infertile by busulfan. The recipients were given a second dose of busulfan to deplete endogenous germ cells and enrich the transplanted cells, and donor cell-derived spermatogenesis was accomplished. This method requires fewer recipients due to higher survival rates, and there is no need to wait for maturation of the founders, which is required when transplanting PGCs into embryos. These results are expected to improve transgenic zebra finch production.

Embryo manipulation in neotropical characiform fish: incubation system, anaesthetic, and PGC transplantation in Prochilodus lineatus.

Primordial germ cells transplantation is a unique approach for conservation and reconstitution of endangered fish species. This study aimed to establish techniques to culture dechorionated embryos in different incubation systems and also to determine anaesthetic concentration for fish recipients in the larval stage for subsequent primordial germ cell transplantation. Intact and dechorionated embryos were divided into three incubation systems: (1) a control group with manual replacement of the solution; (2) a closed environment with high oxygen with manual replacement of the solution; and (3) constant solution recirculation. This combination resulted in six treatments. For the evaluation of anaesthetics for larvae, the concentrations evaluated were 19.5 mM, 24.4 mM, 29.3 mM, and 34.2 mM of 2-phenoxyethanol. Anaesthesia concentration and recovery at different stages were evaluated. For transplantation, primordial germ cells of Astyanax altiparanae were transplanted into anaesthetised larvae (1 dph) of Prochilodus lineatus. Better results were obtained in the recirculation system for dechorionated embryos of P. lineatus for hatching (54.18%) and normal morphology (50.06%). The 2-phenoxyethanol anaesthetic with a dose of 29.3 mM resulted in shorter induction times, in addition to the recovery time between 5 and 10 min. By using this anaesthetic concentration at transplantation, GFP-positive cells were seen in two recipients, but the cells did not proliferate. This study established an effective incubation system for the development of the dechorionated embryo and determined an effective anaesthetic concentration for P. lineatus larvae. In addition, micromanipulation and transplantation of primordial germ cells in neotropical species were conducted for the first time.

RETRACTED ARTICLE: Production of viable chicken by allogeneic transplantation of primordial germ cells induced from somatic cells

The allogeneic transplantation of primordial germ cells (PGCs) derived from somatic cells overcomes the limitation of avian cloning. Here, we transdifferentiate chicken embryo fibroblasts (CEFs) from black feathered Langshan chickens to PGCs and transplant them into White Plymouth Rock chicken embryos to produce viable offspring with characteristics inherited from the donor. We express Oct4/Sox2/Nanog/Lin28A (OSNL) to reprogram CEFs to induced pluripotent stem cells (iPSCs), which are further induced to differentiate into PGCs by BMP4/BMP8b/EGF. DNA demethylation, histone acetylation and glycolytic activation elevate the iPSC induction efficiency, while histone acetylation and glycolytic inhibition facilitate PGCs formation. The induced PGCs (iPGCs) are transplanted into the recipients, which are self-crossed to produce 189/509 somatic cells derived chicken with the donor’s characteristics. Microsatellite analysis and genome sequencing confirm the inheritance of genetic information from the donor. Thus, we demonstrate the feasibility of avian cloning from somatic cells.

Primordial germ cells isolated from individual embryos of red junglefowl and indigenous pheasants of Thailand

Interspecific germline chimerism mediated by transplantation of primordial germ cells (PGCs) of wild species to domestic hosts promises the conservation of wild birds. Cryopreservation of avian eggs and embryos is impracticable, and currently only frozen PGCs enable conservation of both the male and female descendants. Purebred offspring have been obtained from germline chimeras of wild avian species, proving the feasibility of such technology. In vitro propagation has been optimized for avian PGCs of domestic species; however, evidence is rather limited for successful isolation as well as long-term culture from a single embryo of wild species. With accelerating biodiversity loss, we have committed to preserving current genetic resources by freezing PGCs isolated from individual embryos in addition to their genetic material. We have devised a reliable protocol for the isolation and proliferation of PGCs from wild fowls in the family Phasianidae that are conserved in captive breeding (red junglefowl, bar-tailed pheasant, kalij pheasant, Siamese fireback pheasant, and silver pheasant). We obtained individual isolates of cultured circulating PGCs (49.7%, 79/155) as well as tissue PGCs (92.9%, 144/155). The specific co-culture conditions of autologous embryonic cells, without additional growth factors, facilitated the proliferation of so-called tissue PGCs (the remaining PGCs in embryonic tissue following blood aspiration). Only circulating PGCs left in blood vessels and of PGCs migrating to developing gonads have been previously reported. However, the present study is the first to report on the harvest of ectopic PGCs. The defined conditions sustained continuous proliferation of tissue PGCs for at least six months and maintained PGC identity following cryopreservation. Cultured tissue PGCs of these wild species were extensively characterized for their expression of the germ cell-specific proteins, chicken vasa homolog (CVH) and deleted in azoospermia-like (DAZL), as well as the ability to colonize chicken embryonic gonads. The novel protocol is practical for generating enough PGCs for cryopreservation, transplantation, and additionally, it enables isolation of PGCs from both blood circulation and embryonic tissue simultaneously. For conservation purposes, this approach is potentially applicable more widely to other non-domestic birds than those in the family Phasianidae that were investigated in the present study.

Mtor inhibition by INK128 extends functions of the ovary reconstituted from germline stem cells in aging and premature aging mice.

Stem cell transplantation has been generally considered as promising therapeutics in preserving or recovering functions of lost, damaged, or aging tissues. Transplantation of primordial germ cells (PGCs) or oogonia stem cells (OSCs) can reconstitute ovarian functions that yet sustain for only short period of time, limiting potential application of stem cells in preservation of fertility and endocrine function. Here, we show that mTOR inhibition by INK128 extends the follicular and endocrine functions of the reconstituted ovaries in aging and premature aging mice following transplantation of PGCs/OSCs. Follicular development and endocrine functions of the reconstituted ovaries by transplanting PGCs into kidney capsule of the recipient mice were maintained by INK128 treatment for more than 12 weeks, in contrast to the controls for only about 4 weeks without receiving the mTOR inhibitors. Comparatively, rapamycin also can prolong the ovarian functions but for limited time. Furthermore, our data reveal that INK128 promotes mitochondrial function in addition to its known function in suppression of immune response and inflammation. Taken together, germline stem cell transplantation in combination with mTOR inhibition by INK128 improves and extends the reconstituted ovarian and endocrine functions in reproductive aging and premature aging mice.

Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted primordial germ cells

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rate during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cell (PGC) transplantation (PGCT) to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. Firstly, we optimized the procedure for CRISPR/Cas9 targeted PGCT by increasing the efficiencies of genome mutation in PGCs and induction of PGC fates in donor embryos for PGCT. Secondly, the optimized CRISPR/Cas9 targeted PGCT was utilized for generation of maternal-zygotic (MZ) mutants of tcf7l1a (gene essential for head development), pou5f3 (gene essential for zygotic genome activation) and chd (gene essential for dorsal development) at F1 generation with relatively high efficiency. Finally, we revealed some novel phenotypes in MZ mutants of tcf7l1a and chd, as MZtcf7l1a showed elevated neural crest development while MZchd had much severer ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish. It is also feasible to speed up the genome editing in commercial fishes by utilizing a similar approach by surrogate production of CRISPR/Cas9 targeted germ cells.

Conservation of chicken male germline by orthotopic transplantation of primordial germ cells from genetically distant donors†.

Successful derivation and cultivation of primordial germ cells (PGCs) opened the way to efficient transgenesis and genome editing in the chicken. Furthermore, implantation of male PGCs from non-chicken galliform species into the chicken embryos resulted in cross-species germline chimeras and viable offspring. We have recently improved the PGC technology by demonstrating that chicken male PGCs transplanted into the testes of adult cockerel recipients mature into functional sperms. However, the availability of this orthotopic transplantation for cross-species transfer remains to be explored. Here we tested the capacity of genetically distant male PGCs to mature in the microenvironment of adult testes. We derived PGCs from the Chinese black-bone Silkie and transplanted them into infertile White Leghorn cockerels. Within 15-18 weeks after transplantation, we observed restoration of spermatogenesis in recipient cockerels and production of healthy progeny derived from the transplanted PGCs. Our findings also indicate the possibility of cross-species orthotopic transplantation of PGCs. Thus, our results might contribute to the preservation of endangered avian species and maintaining the genetic variability of the domestic chicken.

Preparation of a fish embryo for micromanipulation: staging of development, removal of the chorion and traceability of PGCs in Prochilodus lineatus.

The transplantation of primordial germ cells (PGCs) is a valuable tool for gene-banking and reconstitution by means of a germline chimera. For this technology, studies regarding developmental stages and traceability of PGCs are necessary. The objective of this study was to develop a micromanipulation procedure for the future establishment of cryobanks of PGCs in migratory characins. Incubation temperatures were evaluated at 22 ° C, 26 ° C, and 30 ° C in order to synchronize developmental stages. The highest hatching rates and the lowest abnormality rate arose at 26° C, which was considered to be the best incubation temperature. Enzymatic removal of the chorion was determined to be best using 0.05% pronase, in which the embryos presented better survival rates. In order to visualize PGCs in vivo, artificial GFP-nos1 3'UTR mRNA was injected and the migration route was observed in vivo as PGCs were visualized firstly at the segmentation stage (6 to 13 somites). The number of GFP positive cells ranged from 8 to 20 per embryo (mean of 13.8; n = 5). After hatching, GFP-positive cells increased to 14 to 27 embryos (mean of 19.8; n = 5). Visualization of the GFP-positive cells was possible at 10 days post hatching, and at this stage, the cells were positioned in the yolk extension region. This is the first report on PGC visualization in vivo in Neotropical fish; the obtained data provide information on the identification and migration of PGCs. The information presented in this work brings new insights in gene banking in Neotropical species and subsequent reconstitution through a germinal germline chimera.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus.

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to "cut and paste" embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying "leapfrog transplants" (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Reconstitution of ovarian function following transplantation of primordial germ cells

Ovarian aging occurs earlier than somatic aging. We tested the hypothesis that ovarian functions could be artificially reconstructed by transplantation of primordial germ cells (PGCs). We compared various methods for transplantation of PGCs aggregated with gonadal somatic cells and showed that reconstituted ovaries exhibited folliculogenesis after transplantation of PGCs-aggregates into either kidney capsule or ovarian bursa. Neo-oogenesis occurred early after transplantation, as evidenced by the presence of prophase I meiocytes displaying homologous pairing. Moreover, endocrine function was recovered in ovariectomized recipients, including elevated levels of AMH and estradiol. Interestingly, folliculogenesis in the reconstituted ovaries failed to sustain past four weeks. Regardless of transplantation method, follicles diminished after 45 days, accompanied by increased apoptosis, and were undetectable after two months. Meanwhile, no replicative PGCs or prophase I meiocytes could be found. Together, transplantation of PGCs can effectively reconstitute ovarian functions but for limited time. These data suggest that PGCs do not undergo self-renewal but rapidly enter meiosis following transplantation. Global activation of primordial follicles in artificial ovaries can result in further rapid loss of germ cells. Methods for maintaining self-renewal and expansion in vivo of PGCs and controlling follicle activation will be essential for continuing maintenance of the functional reconstructed ovaries.

Avian Biotechnology.

Primordial germ cells (PGCs) generate new individuals through differentiation, maturation and fertilization. This means that the manipulation of PGCs is directly linked to the manipulation of individuals, making PGCs attractive target cells in the animal biotechnology field. A unique biological property of avian PGCs is that they circulate temporarily in the vasculature during early development, and this allows us to access and manipulate avian germ lines. Following the development of a technique for transplantation, PGCs have become central to avian biotechnology, in contrast to the use of embryo manipulation and subsequent transfer to foster mothers, as in mammalian biotechnology. Today, avian PGC transplantation combined with recent advanced manipulation techniques, including cell purification, cryopreservation, depletion, and long-term culture in vitro, have enabled the establishment of genetically modified poultry lines and ex-situ conservation of poultry genetic resources. This chapter introduces the principles, history, and procedures of producing avian germline chimeras by transplantation of PGCs, and the current status of avian germline modification as well as germplasm cryopreservation. Other fundamental avian reproductive technologies are described, including artificial insemination and embryo culture, and perspectives of industrial applications in agriculture and pharmacy are considered, including poultry productivity improvement, egg modification, disease resistance impairment and poultry gene "pharming" as well as gene banking.

Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes.

CRISPR/Cas9 genome editing is revolutionizing genetic loss-of-function analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1 heterozygotes. Phenotypic analysis occurs in the F2 generation following F1 intercrosses. Thus, mutant analyses will require multi-generational studies. Second, when targeting essential genes, efficient mutagenesis of founders is often lethal, preventing the acquisition of mature animals. Reducing mutagenesis levels may improve founder survival, but results in lower, more variable rates of germline transmission. Therefore, an efficient approach to study lethal mutations would be useful. To overcome these shortfalls, we introduce 'leapfrogging', a method combining efficient CRISPR mutagenesis with transplantation of mutated primordial germ cells into a wild-type host. Tested using Xenopus tropicalis, we show that founders containing transplants transmit mutant alleles with high efficiency. F1 offspring from intercrosses between F0 animals that carry embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an entire generation of breeding. We anticipate that leapfrogging will be transferable to other species.

Poultry genetic resource conservation using primordial germ cells.

The majority of poultry genetic resources are maintained in situ in living populations. However, in situ conservation of poultry genetic resources always carries the risk of loss owing to pathogen outbreaks, genetic problems, breeding cessation, or natural disasters. Cryobanking of germplasm in birds has been limited to the use of semen, preventing conservation of the W chromosome and mitochondrial DNA. A further challenge is posed by the structure of avian eggs, which restricts the cryopreservation of ova and fertilized embryos, a technique widely used for mammalian species. By using a unique biological property and accessibility of avian primordial germ cells (PGCs), precursor cells for gametes, which temporally circulate in the vasculature during early development, an avian PGC transplantation technique has been established. To date, several techniques for PGC manipulation including purification, cryopreservation, depletion, and long-term culture have been developed in chickens. PGC transplantation combined with recent advanced PGC manipulation techniques have enabled ex situ conservation of poultry genetic resources in their complete form. Here, the updated technologies for avian PGC manipulation are introduced, and then the concept of a poultry PGC-bank is proposed by considering the biological properties of avian PGCs.

Generation of mouse functional oocytes in rat by xeno-ectopic transplantation of primordial germ cells.

Primordial germ cells (PGCs) are germ cell progenitors in the fetal genital ridge; female PGCs give rise to definitive oocytes that contribute to the next generation. Artificial PGCs have been induced in vitro from pluripotent stem cells and gonad-like tissue has been induced in vivo by cotransplantation of PGCs with PGC-free gonadal cells. To apply these technologies to human infertility treatment or conservation of rare species, PGC transplantation must be established in xenogenic animals. Here, we established a xenogeneic transplantation model by inducing ovary-like tissue from PGCs in xenogenic animals. We transplanted enzymatically dispersed PGCs with PGC-free gonadal cells under the kidney capsule of xenogenic immunodeficient animals. The transplanted cells formed ovary-like tissues under the kidney capsule. These tissues were histologically similar to the normal gonad and expressed the oocyte markers Vasa and Stella. In addition, mouse germinal vesicle-stage oocyte-like cells collected from ovary-like tissue in rats matured to metaphase II via in vitro maturation and gave rise to offspring by intracytoplasmic sperm injection. Our studies show that rat/mouse female PGCs and PGC-free gonadal cells can develop and reconstruct ovary-like tissue containing functional oocytes in an ectopic xenogenic microenvironment.

Sexually Dimorphic Expression of vasa Isoforms in the Tongue Sole (Cynoglossus semilaevis)

The vasa gene encodes an ATP-dependent RNA helicase of the DEAD box protein family that functions in a broad range of molecular events involving duplex RNA. In most species, the germline specific expression of vasa becomes a molecular marker widely used in the visualization and labeling of primordial germ cells (PGCs) and a tool in surrogate broodstock production through PGC transplantation. The vasa gene from tongue sole (Cynoglossus semilaevis) was characterized to promote the development of genetic breeding techniques in this species. Three C. semilaevis vasa transcripts were isolated, namely vas-l, vas-m, and vas-s. Quantitative real-time PCR results showed that C. semilaevis vasa transcripts were prevalently expressed in gonads, with very weak expression of vas-s in other tissues. Embryonic development expression profiles revealed the onset of zygotic transcription of vasa mRNAs and the maternal deposit of the three transcripts. The genetic ZW female juvenile fish was discriminated from genetic ZZ males by a pair of female specific primers. Only the expression of vas-s can be observed in both sexes during early gonadal differentiation. Before PGCs started mitosis, there was sexually dimorphic expression of vas-s with the ovary showing higher levels and downward trend. The results demonstrated the benefits of vasa as a germline specific marker for PGCs during embryonic development and gonadal differentiation. This study lays the groundwork for further application of C. semilaevis PGCs in fish breeding.

The Origin And Migration Of Primordial Germ Cells In Sturgeons

Primordial germ cells (PGCs) arise elsewhere in the embryo and migrate into developing gonadal ridges during embryonic development. In several model animals, formation and migration patterns of PGCs have been studied, and it is known that these patterns vary. Sturgeons (genus Acipenser) have great potential for comparative and evolutionary studies of development. Sturgeons belong to the super class Actinoptergii, and their developmental pattern is similar to that of amphibians, although their phylogenetic position is an out-group to teleost fishes. Here, we reveal an injection technique for sturgeon eggs allowing visualization of germplasm and PGCs. Using this technique, we demonstrate that the PGCs are generated at the vegetal pole of the egg and they migrate on the yolky cell mass toward the gonadal ridge. We also provide evidence showing that PGCs are specified by inheritance of maternally supplied germplasm. Furthermore, we demonstrate that the migratory mechanism is well-conserved between sturgeon and other remotely related teleosts, such as goldfish, by a single PGCs transplantation (SPT) assay. The mode of PGCs specification in sturgeon is similar to that of anurans, but the migration pattern resembles that of teleosts.

Solea senegalensis vasa transcripts: molecular characterisation, tissue distribution and developmental expression profiles

The Vasa protein is an RNA helicase belonging the DEAD (Asp-Glu-Ala-Asp)-box family. The crucial role played by the vasa gene in the germ-cell lineage of both vertebrates and invertebrates has made this gene a useful molecular marker for germinal cells and a useful tool in surrogate broodstock production using primordial germ cell transplantation. With the aim of establishing a novel approach to improving Solea senegalensis broodstock management, the vasa gene in this species was characterised. Four S. senegalensis vasa transcripts were isolated: Ssvasa1, Ssvasa2, Ssvasa3 and Ssvasa4. Their phylogenetic relationship with other vasa homologues was determined confirming the high degree of conservation of this helicase throughout evolution. Our qPCR results showed that S. senegalensis vasa transcripts are prevalently expressed in gonads, with ovary-specific expression for Ssvasa3 and Ssvasa4. During embryonic and larval development, a switch between the longest and the shortest transcripts was observed. While Ssvasa1 and Ssvasa2 were maternally supplied, Ssvasa3 and Ssvasa4 depended on the de novo expression program of the growing juveniles, suggesting that vasa mRNA could be involved in Senegalese sole gonad differentiation. In situ hybridisation and immunohistochemical analysis performed in 150-days after hatching (DAH) larvae showed vasa product expression in the germinal region of early gonads. In our work we demonstrated the usefulness of Ssvasa mRNAs as molecular markers for primordial germ cells and germinal cells during embryonic development, larval ontogenesis and gonad differentiation. Furthermore, our results confirmed the potential of vasa to help investigate germinal cell biotechnology for Senegalese sole reproduction.

Biological Characteristics of Fish Germ Cells and their Application to Developmental Biotechnology

We have revealed several unique characteristics of germ cell development using rainbow trout, including the fact that spermatogonia transplanted into the peritoneal cavity of newly hatched embryos migrate toward recipient gonads, that spermatogonia transplanted into female recipients start oogenesis and produce functional eggs and that diploid germ cells transplanted into triploid trout can complete gametogenesis. By combining these unique features of fish germ cells, we established allogeneic and xenogeneic transplantation systems for spermatogonia in several fish species. Spermatogonia isolated from the mature testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout were transplanted into the peritoneal cavity of triploid masu salmon newly hatched embryos. These spermatogonia migrated toward recipient salmon genital ridges with extending pseudopodia and were subsequently incorporated into them. We further confirmed that the donor-derived spermatogonia resumed gametogenesis and produced sperm and eggs in male and female salmon recipients, respectively. By inseminating the resulting eggs and sperm, we obtained only rainbow trout offspring in the F1 generation, suggesting that the triploid salmon recipients produced functional gametes derived only from donor trout. We further confirmed that this intra-peritoneal transplantation of germ cells is applicable to several marine fishes, which could be of benefit in the production of bluefin tuna that has a large broodstock (>100 kg) and is difficult to maintain in captivity. Gamete production of bluefin tuna could be more easily achieved by generating a surrogate species, such as mackerel, that can produce tuna gametes.

Inter-species transplantation and migration of primordial germ cells in cyprinid fish

Primordial germ cells (PGCs) are the only cells in developing embryos that can transmit genetic information to the next generation. PGCs therefore have considerable potential value for gene banking and cryopreservation, particularly via production of donor gametes using germ-line chimeras. In some animal species, including teleost fish, the feasibility of using PGC transplantation to obtain donor-derived offspring, within and between species, has been demonstrated. Successful use of PGC transplantation to produce germ-line chimeras is absolutely dependent on the migration of the transplanted cells from the site of transplantation to the host gonadal region. Here, we induced germ-line chimeras between teleost species using two different protocols: blastomere transplantation and single PGC transplantation. We evaluated the methods using the rate of successful migration of transplanted PGCs to the gonadal region of the host embryo. First, we transplanted blastomeres from zebrafish, pearl danio, goldfish, or loach into blastula-stage zebrafish embryos. Some somatic cells, derived from donor blastomeres, were co-transplanted with the PGCs and formed aggregates in the host embryos; a low efficiency of PGC transfer was achieved. Second, a single PGC from the donor species was transplanted into a zebrafish embryo. In all inter-species combinations, the donor PGC migrated toward the gonadal region of the host embryo at a comparatively high rate, regardless of the phylogenetic relationship of the donor and host species. These transplantation experiments showed that the mechanism of PGC migration is highly conserved beyond the family barrier in fish and that transplantation of a single PGC is an efficient method for producing inter-species germ-line chimeras.