Transfer Of Primordial Germ Cells Research Articles

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.

The potential for archiving and reconstituting valuable strains of turkey (Meleagris gallopavo) using primordial germ cells

Diseases such as avian influenza can destroy turkey flocks, potentially resulting in the loss of valuable or rare genetic material. Consequently, there is an urgent need to develop a means to archive such germplasm. Germline chimeras produced by intravascular transfer of primordial germ cells (PGC) have been reported in other avian species but not turkeys. This study examined the feasibility of both establishing an archive of frozen PGC, and producing germline chimeras by injecting the thawed PGC into host embryos. To meet these aims, the following experiments were performed: (1) PGC identification within turkey embryos; (2) development of an efficient method for isolation of turkey PGC; (3) demonstration that PGC can be cryopreserved, recovered, and retain viability; (4) reinjection into embryos and detection of injected PGC. Primordial germ cells were identified using periodic acid-Schiff reagent and the immunological marker OLP-1. Bloodstream PGC were isolated using Ficoll density gradient centrifugation with PGC recovery peaking at stages 13, 14, and 15 with 32 ± 4.9, 33 ± 6.4, and 26 ± 5.4 PGC recovered, respectively. Primordial germ cells were frozen using Dulbecco's modified Eagle medium, 20% fetal calf serum, and 10% dimethylsulfoxide and demonstrated 90 ± 1.7% viability after 3 mo frozen in liquid nitrogen. Freshly isolated and frozen thawed DiI- and Q-Tracker-labeled PGC repopulated stage 30 gonads after vascular transfer into ex ovo cultured embryos. The DiI-labeled cells repopulated gonads less frequently, with 36 ± 13.2% of gonads containing the DiI-labeled PGC, and 7 ± 3.8% of reinjected PGC reaching the gonads of positive embryos. The Q-tracker-labeled cells were detected more frequently in embryos, with 67 ± 21.1% having positive signals, and 44 ± 4.9% of reinjected Q-tracker-labeled PGC colonized the gonads of positive embryos. This study demonstrated the feasibility of using turkey PGC to archive turkey germplasm from different strains because frozen PGC reintroduced into host embryos can colonize the host gonads, suggesting the possibility of producing turkey germline chimeras.

Production of Pure Hinai-dori with Normal Reproductive Capability from Transferred Primordial Germ Cells

The production method of germline chimeras by transfer of primordial germ cells (PGCs) can be used in gene banking of chickens. However, little is known about the reproductive capability of offspring obtained by mating of male and female germline chimeras. To cryopreserve chicken genetic resources at the cellular level in future, it is necessary to demonstrate whether offspring originating from donor-derived PGCs have normal reproductive capability. In this study, we attempted to obtain the pure Hinai-droi offspring by mating of male and female germline chimeras produced by the transplantation of Hinai-dori PGCs into the Akita White Leghorn line (WL) embryos. Hinai-dori offspring from germline chimeras were screened using the Hinai-dori-specific autosomal microsatellite marker, namely, ADL0315, which allows distinction between the Hinai-dori breed and Akita WL. After sexual maturity of the Hinai-dori offspring originating from donor-derived Hinai-dori PGCs, the reproductive capability (egg production rate, fertilization rate and hatchability) was examined. Percentage hen-day egg production was investigated between 21 and 60 wk of age, and fertilization rate and hatchability were investigated between 43 and 60 wk of age, respectively. Among the 105 progeny obtained by mating of male and female germline chimeras, a single brown-colored female hatchling was obtained. The results from molecular genotyping using the Hinai-dori-specific microsatellite marker ADL0315 were consistent with results from morphological identification, and the brown-colored female hatchling was identified as the pure Hinai-dori breed. The mean percentage hen-day egg production, fertilization rate and hatchability of the Hinai-dori hen were 36.4%, 58.1% and 53.2%, which were similar to those of normal Hinai-dori hens. These results indicated that the pure Hinai-dori offspring originating from donor-derived Hinai-dori PGCs has normal reproductive capability.

상업용 동결보호제를 이용한 한국재래닭(오계) 원시생식세포의 동결 보존

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the unique anatomical and physiological characteristics of the avian egg. Thus an alternative strategy for conservation of oviparous species of animals must be developed. Recent technological developments for producing germline chimeras by the transfer of primordial germ cells (PGCs) into recipient embryos has enabled the conservation and retrieval of chicken genetic resources in their complete form. In the present study, fertilized eggs were incubated for about 5.5 days to obtain embryos at stage 28. The whole embryo was collected from the germinal gonad using a fine glass micro pipette under a microscope. The PGCs were then purified using MACS method. Two commercially available cryoprotectants (A and B) were used to preserve the PGCs, and EG were used as a control. The average recovery rate of PGCs after thawing was 35.5% and 60.5% with the A and B treatments, respectively. There was no significant difference between B treatments and control, which showed an average recovery rate of 52.8%. However, the recovery rate obtained using A cryoprotectant (35.5%) was significantly lower than using treatment control and B. The average viability of the PGCs after thawing were 77.9% and 77.4% for cryoprotectants A and B, respectively, and the control were was 81.6%. There was no statistically significant difference between the two treatments and control. It was concluded that all of the available cryoprotectants examined in this study could be used for preservation of PGCs from embryos. Further experiments to produce germline chimera from PGCs preserved using this techniques are strongly recommended.

Development, differentiation and manipulation of chicken 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.

Identification of Germline Chimeric Chickens Produced by Transfer of Primordial Germ Cells Using a Hinai-dori-specific Microsatellite Marker

The production of germline chimeric chickens by transfer of primordial germ cells (PGCs) is an effective technique for the preservation and regeneration of genetic resources in chickens. To date, the most widely used method to identify germline chimeric chickens is a testcross using differences in plumage color between the donor and recipient breeds. However, this method is time consuming and laborious. Therefore, simple high-precision molecular techniques for the identification of germline chimeric chickens need to be developed. In this study, we verified the practicability of using a Hinai-dori-specific microsatellite marker that was previously developed to identify germline chimeric chickens. A Hinai-dori-specific microsatellite marker ABR0633 can distinguish the Hinai-dori breed from White Leghorn (WL); thus, these two breeds were used as donor and recipient embryos, respectively, in this study. PGCs obtained from embryonic gonads of Hinai-dori were micro-injected into either the subgerminal cavity or dorsal aorta of WL recipient embryos to produce germline chimeric chickens. A portion of both left and right testes was removed from the manipulated male chicks and was used for genotyping to verify the possibility of identifying germline chimeric chickens. Semen was then obtained from matured manipulated male chickens and was used for genotyping. Simultaneously, these chickens were crossed with Hinai-dori females by artificial insemination to produce offspring. No donor-derived Hinai-dori allele was detected in the testicular tissues from the manipulated male chicks. However, donor-derived Hinai-dori allele was detected in the semen from two manipulated male chickens (2/2) that were produced by microinjection of Hinai-dori PGCs into the dorsal aorta. In the progeny tests, Hinai-dori PGC-derived offspring were obtained from these two chickens. Moreover, only the donor-derived Hinai-dori allele was detected in the offspring that were judged as the Hinai-dori breed by plumage color. Therefore, the offspring were confirmed as the Hinai-dori breed both phenotypically and genotypically. We conclude that a Hinai-dori-specific microsatellite marker is suitable to identify germline chimeric chickens and the use of this method could reduce the time and labor needed for testcrosses.

Germline Replacement by Transfer of Primordial Germ Cells into Partially Sterilized Embryos in the Chicken1

We report a novel technique for almost complete replacement of the recipient germline with donor germ cells in the chicken. Busulfan solubilized in a sustained-release emulsion was injected into the yolk of fertile eggs before incubation. A dose of 100 microg was found to provide the best outcome in terms of reducing the number of endogenous primordial germ cells (PGCs) in embryonic gonads (0.6% of control numbers) and hatchability (36.4%). This was applied for preparing partially sterilized embryos to serve as recipients for the transfer of exogenous PGCs. Immunohistochemical analysis showed that the proportion of donor PGCs in busulfan-treated embryos was significantly higher than in controls (98.6% vs. 6.4%). Genetic cross-test analysis revealed that the germline transmission rate in busulfan-treated chickens was significantly higher than in controls (99.5% vs. 6.0%). Of 11 chimeras, 7 produced only donor-derived progenies, suggesting that these produced only donor-derived gametes in the recipient's gonads. This novel germline replacement technique provides a powerful tool for studying germline differentiation, for generating transgenic individuals, and for conserving genetic resources in birds.

The reversible developmental unipotency of germ cells in chicken

We recently developed bimodal germline chimera production approaches by transfer of primordial germ cells (PGCs) or embryonic germ cells (EGCs) into embryos and by transplantation of spermatogonial stem cells (SSCs) or germline stem cells (GSCs) into adult testes. This study was undertaken to investigate the reversible developmental unipotency of chicken germ cells using our established germline chimera production systems. First, we transferred freshly isolated SSCs from adult testis or in vitro cultured GSCs into stage X and stage 14-16 embryos, and we found that these transferred SSCs/GSCs could migrate to the recipient embryonic gonads. Of the 527 embryos that received SSCs or GSCs, 135 yielded hatchlings. Of 17 sexually mature males (35.3%), six were confirmed as germline chimeras through testcross analysis resulting in an average germline transmission efficiency of 1.3%. Second, PGCs/EGCs, germ cells isolated from embryonic gonads were transplanted into adult testes. The EGC transplantation induced germline transmission, whereas the PGC transplantation did not. The germline transmission efficiency was 12.5 fold higher (16.3 vs 1.3%) in EGC transplantation into testis (EGCs to adult testis) than that in SSC/GSC transfer into embryos (testicular germ cells to embryo stage). In conclusion, chicken germ cells from different developmental stages can (de)differentiate into gametes even after the germ cell developmental clock is set back or ahead. Use of germ cell reversible unipotency might improve the efficiency of germ cell-mediated germline transmission.

Expression of mRNA for 3HADH in manipulated embryos to produce germline chimeric chickens.

Germline chimeric chickens were produced by the transfer of primordial germ cells (PGCs) or blastoderm cells. The hatchability of eggs produced by transfer of exogenous PGCs is usually low. The purpose of the present study was investigated to express (3-hydroxyacyl CoA dehydrogenase) 3HADH which is a limiting enzyme in the beta-oxidation of fatty acids for hatching energy. Manipulations of both donor and recipient eggshells were as follows. A window approximately 10 mm in diameter was opened at the pointed end of the eggs at stage 12-15 days incubation. Donor PGCs, taken from the blood vessels of donor embryos from fertilized eggs at the same stage of development, were injected into the blood vessels of recipient embryos. The muscles of chicks in the eggs with transferred PGCs were removed after 20 days of incubation. A cDNA was prepared from the total RNA. The expression of 3HADH in the manipulated embryos was investigated using real-time PCR analysis. Real-time PCR analysis showed that expression of 3HADH was reduced in the muscles of manipulated embryos.

Depletion of Primordial Germ Cells (PGCs) by X-irradiation to Extraembryonic Region of Chicken Embryos and Expression of Xenotransplanted Quail PGCs

The generation of germline chimeras by the transfer of primordial germ cells (PGCs) requires incorporation of the PGCs of the donor into the gonadal tissue of the recipient embryo. We investigated the utility of soft x-irradiation with application of a lead (12×3×0.25mm, ∼0.1g) shield to the embryo proper for the production of chicken-quail germline chimeras. Chicken embryos shielded during irradiation for 120 s (∼7.2Gy) at stages 13 to 17 showed a hatchability of 35% (106/301), whereas the hatchability of unshielded embryos was 26% (27/105). The relative population of gonadal PGCs at stage 30 for embryos irradiated at stage 13 with or without shielding was 13 and 5%, respectively, of the value for nonirradiated controls. Chicken embryos irradiated at stages 13 or 14 with or without shielding and transfused with quail embryonic blood containing PGCs each exhibited ∼130 relative population of donor PGCs in the left gonad at stage 30. Xenotransplanted hatchlings exhibited donor-derived PGCs as detected by Southern hybridization and PCR. Exposure of chicken embryos to ∼7.2Gy of x-radiation at stage 13 with the application of a lead shield to the embryo proper is thus a feasible approach to depletion of endogenous germ cells and the production of chicken-quail germline chimeras.

Transfer of blood containing primordial germ cells between chicken eggs development of embryonic reproductive tract.

The present study was carried out to investigate development of recipient chicken embryonic reproductive tracts which are transferred chicken primordial germ cells (PGCs). It is thought that differentiation of PGCs is affected by the gonadal somatic cells. When female PGCs are transferred to male embryos, it is possible that they differentiate to W-spermatogonia. However, the relationship development between PGCs and gonads has not been investigated. At stage 12-15 of incubation of fertilized eggs, donor PGCs, which were taken from the blood vessels of donor embryos, were injected into the blood vessels of recipient embryos. The gonads were removed from embryos that died after 16 days of incubation and from newly hatched chickens and organs were examined for morphological and histological features. The survival rate of the treated embryos was 13.6% for homo-sexual transfer of PGCs (male PGCs to male embryo or female PGCs to female embryo) and 28.9% for hetero-sexual transfer PGCs (male PGCs to female embryo or female PGCs to male embryo) when determined at 15 days of incubation. The gonads of embryos arising from homo-sexual transfer appeared to develop normally. In contrast, embryos derived from hetero-sexual transfer of PGCs had abnormal gonads as assessed by histological observation. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.

Effects of Soft X-Ray Irradiation to Stage X Blastoderm on Restriction of Proliferation of Primordial Germ Cells in Early Chicken Embryos

It is necessary to deplete the endogenous primordial germ cells (PGCs) from recipient embryos when germline chimaeric chickens are produced by the transfer of PGCs. In this study, effects of soft X-ray irradiation on the restriction of PGC proliferation were examined to obtain recipient embryos with as many depleted PGCs as possible. The stage X blastoderm was irradiated with soft X-rays (15 kVP, 5mA, 0.083nm, 16 cm distance) for 20-180 seconds (0.27-2.4Gy), and concentrations of PGCs in the bloodstream of embryos at stages 13-16 were analysed. The viabilities of embryos irradiated with soft X-rays for 20-90 seconds was not significantly different from those in the control, but were markedly reduced when embryos were irradiated with soft X-rays for 120-180 seconds. The concentration of PGCs in the bloodstream of embryos was reduced by increasing the irradiation time of soft X-rays. In particular, the concentration of PGCs at stages 14-15 started to decrease rapidly when irradiated with soft X-rays for more than 90 seconds. Administration of busulfan in the stage X blastoderm showed a similar effect to soft X-ray irradiation on the concentration of PGCs in the bloodstream of embryos. Our results suggest that a 90-second irradiation of soft X-rays to the stage X blastoderm is recommended for reducing the concentration of PGCs in chicken embryos without affecting their viability.

Genetic manipulation in chickens

Development of chicken embryo culture techniques provides precise methods for the manipulation of early chicken embryos and has made it possible to analyse the results of the manipulation in hatched chickens. Various attempts have been made to introduce exogenous DNA into the chicken germline, but an efficient method for producing transgenic chickens by non-viral methods has not yet been devised. Since the system for producing germline chimaeric chickens has been established by the transfer of primordial germ cells isolated from early stage embryos, manipulation of primordial germ cells seems to be one of the most promising methods for genetic manipulation in chickens.

Foreign Gene Expression in the Gonads of Chimaeric Chicken Embryos by Transfer of Primordial Germ Cells Transfected in vitro by Lipofection for 24 Hours

Foreign Gene Expression in the Gonads of Chimaeric Chicken Embryos by Transfer of Primordial Germ Cells Transfected in vitro by Lipofection for 24 Hours

Inter embryonic (homo- or hetero-sexual) transfer of primordial germ cells between chicken embryos.

Chicken primordial germ cells (PGCs) collected from thecirculating blood in embryonic vessels at stage 13-15 were inter-embryonically, homo- or hetero-sexually,transferred to the blood vessels of recipient embryosat the same stage of development. Approximately 30%of the embryos treated with hetero-sexual transfer of PGCs had abnormal gonads, showing ovotestis likeorgans. In this case, some of these reversed gonadswere considered to be dependent upon the ratio of thenumber of PGCs from donor to recipient embryos. Oneof the treated embryos possessed completely reversedorgans. Therefore, the introduction of exogenousembryonic vessels was thought to be also useful forproducing transgened gonads.

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 isolated from embryonic blood.

This study was carried out to elucidate whether primordial germ cells, obtained from embryonic blood and transferred into partially sterilized male and female recipient embryos, could differentiate into functional gametes and give rise to viable offspring. Manipulated embryos were cultured until hatching and the chicks were raised until maturity, when they were mated. When the sex of the donor primordial germ cells and the recipient embryo was the same, 15 out of 22 male chimaeric chickens (68.2%) and 10 out of 16 female chimaeric chickens (62.5%) produced donor-derived offspring. When the sex of the donor primordial germ cells and the recipient embryo was different, 4 out of 18 male chimaeric chickens (22.2%) and 2 out of 18 female chimaeric chickens (11.1%) produced donor-derived offspring. The rates of donor-derived offspring from the chimaeric chickens were 0.6-40.0% in male donor and male recipient and 0.4-34.9% in female donor and female recipient. However, the rates of donor-derived offspring from the chimaeric chickens were 0.4-0.9% in male donor and female recipient and 0.1-0.3% in female donor and male recipient. The presence of W chromosome-specific repeating sequences was detected in the sperm samples of male chimaeric chickens produced by transfer of female primordial germ cells. These results indicate that primordial germ cells isolated from embryonic blood can differentiate into functional gametes giving rise to viable offspring in the gonads of opposite-sex recipient embryos and chickens, although the efficiency was very low.

Developmental origin of avian primordial germ cells and its unique differentiation in the gonads of mixed-sex chimeras.

Developmental origin of avian primordial germ cells and its unique differentiation in the gonads of mixed-sex chimeras.

Expression of exogenous DNA in the gonads of chimaeric chicken embryos produced by transfer of primordial germ cell transfected in vitro and subsequent fate of the introduced DNA.

The expression and fate of exogenous DNA (lacZ gene), introduced into the gonads of chimaeric embryos and chickens that had been produced by transfer of primordial germ cells (PGCs) transfected in vitro, were examined. PGCs obtained from embryonic blood were transfected in vitro by lipofection and transferred to the partially sterilized recipient embryos. Expression of the lacZ gene was observed in the gonads of chimaeric embryos incubated for 3 days after the PGC injection (71.2%, 37/52). Introduction of the lacZ gene into the gonads of chimaeric embryos was confirmed by PCR analysis. The percentage of embryos with gonads positive for the lacZ gene was 95% (38/40) after 3 days of incubation after the PGC injection. The lacZ gene, however, appeared to persist episomally but was gradually lost during embryonic development. After 17 days of incubation after the PGC injection, the lacZ gene was detected in only 14.3% (3/21) of the embryos examined. Although the lacZ gene was detected in the gonads of two hatched chicks (11.1%), it was not detected in the gonads of chimaeric chickens at sexual maturity. Offspring derived from the lipofected PGCs were obtained from the chimaeric chickens at frequencies of 12.1-69.9% in males and 71.6-97.6% in females. The technique developed in the present work could be used to test the expression of exogenous DNA in the gonads of early chicken embryos and should facilitate the production of transgenic chickens.

Donor primordial germ cell-derived offspring from recipient germline chimaeric chickens: Absence of long term immune rejection and effects on sex ratios

1. Germline chimaeric chickens were produced by the transfer of primordial germ cells, and the generation of donor-derived offspring was examined for a maximum of 146 weeks. 2. The frequencies of donor-derived offspring from the chimaeras were 47% to 97%, and no apparent changes in frequency were observed with increasing age during the test period. 3. Differentiation of donor primordial germ cells into functional gametes appeared to be restricted to a degree at some developmental stage in the gonads of chimaeric chickens of the opposite sex.

Production of donor-derived offspring by transfer of primordial germ cells in Japanese quail.

We transfused concentrated primordial germ cells (PGCs) of the black strain (D: homozygous for the autosomal incomplete dominant gene, D) of quail into the embryos of the wild-type plumage strain (WP: d+/d+) of quail. The recipient quail were raised until sexual maturity and a progeny test of the putative germline chimeras was performed to examine the donor gamete-derived offspring (D/d+). Thirty-one percent (36/115) of the transfused quail hatched and 21 (13 females and 8 males) of them reached maturity. Five females and 2 males were germline chimeras producing donor gamete-derived offspring. Transmission rates of the donor derived gametes in the chimeric females and males were 1.8-8.3% and 2.6-63.0%, respectively. Germline chimeric and the other putative chimeric males were also test-mated with females from the sex-linked imperfect albino strain (AL: d+/d+, al/W, where al indicates the sex-linked imperfect albino gene on the Z chromosome, and W indicates the W chromosome) for autosexing of W-bearing spermatozoa: No albino offspring were born.