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

Nakamura, Yatsuka; Usui, Fumitake; Ono, Tamao; Takeda, Kumiko; Nirasawa, Keijiro; Kono, Hiroshi; Tagami, Takahiro

doi:10.1095/biolreprod.110.083923

Cited by 63 publications

(60 citation statements)

References 37 publications

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“…Chicken embryos in the 9-Gy-irradiated group did not survive to hatching, and this dosage was therefore considered to be lethal when embryos were irradiated at stages 13-15. The hatching rate of nonirradiated control embryos in this study was slightly lower than that in our previous study [15] (69.6 vs. 74.3%), probably due to the temporary drop in temperature.…”

Section: Discussioncontrasting

confidence: 85%

“…This method enables the efficient and stable depletion of endogenous PGCs. When donor PGCs were transferred into recipient embryos treated with busulfan, the germline transmission rate was increased 16.6-fold compared with control, untreated embryos [15]. The production of germline chimeric chickens usually involves the transfer of donor PGCs into the bloodstream of recipient embryos between 48 and 64 h of incubation (stages 13-17: Arabic numerals refer to the staging system of Hamburger and Hamilton [16]).…”

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confidence: 99%

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X-irradiation Removes Endogenous Primordial Germ Cells (PGCs) and Increases Germline Transmission of Donor PGCs in Chimeric Chickens

et al. 2012

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Abstract. Primordial germ cells (PGCs) are embryonic precursors of germline cells with potential applications in genetic conservation, transgenic animal production and germline stem cell research. These lines of research would benefit from improved germline transmission of transplanted PGCs in chimeric chickens. We therefore evaluated the effects of pretransplant X-irradiation of recipient embryos on the efficacy of germline transmission of donor PGCs in chimeric chickens. Intact chicken eggs were exposed to X-ray doses of 3, 6 and 9 Gy (dose rate = 0.12 Gy/min) after 52 h of incubation. There was no significant difference in hatching rate between the 3-Gy-irradiated group and the nonirradiated control group (40.0 vs. 69.6%), but the hatching rate in the 6-Gy-irradiated group (28.6%) was significantly lower than in the control group (P<0.05). No embryos irradiated with 9 Gy of X-rays survived to hatching. X-irradiation significantly reduced the number of endogenous PGCs in the embryonic gonads at stage 27 in a dose-dependent manner compared with nonirradiated controls. The numbers of endogenous PGCs in the 3-, 6-and 9-Gy-irradiated groups were 21.0, 9.6 and 4.6% of the nonirradiated control numbers, respectively. Sets of 100 donor PGCs were subsequently transferred intravascularly into embryos irradiated with 3 Gy X-rays and nonirradiated control embryos. Genetic cross-test analysis revealed that the germline transmission rate in the 3-Gyirradiated group was significantly higher than in the control group (27.5 vs. 5.6%; P<0.05). In conclusion, X-irradiation reduced the number of endogenous PGCs and increased the germline transmission of transferred PGCs in chimeric chickens. Key words: Chicken, Germline chimera, Primordial germ cell, X-ray (J. Reprod. Dev. 58: [432][433][434][435][436][437] 2012) P rimordial germ cells (PGCs) are the founder germline cells. In chickens, PGCs are scattered in the center of the blastodisc of freshly oviposited eggs (stage X: Roman numerals refer to the staging system of Eyal-Giladi and Kochav [1]). Following the formation of the primitive streak, PGCs move passively to the anterior border of the extraembryonic region, the so-called germinal crescent region [2]. They then enter the developing vascular network in the germinal crescent region and are transported by the embryonic circulation to the intermediate mesoderm, where they leave the blood vessels and migrate to the genital ridge [3,4]. After settling in the gonads, the PGCs proliferate and then differentiate into functional gametes.A technique for producing live offspring from isolated PGCs following their intravascular transplantation into developing embryos was initially established in chickens in 1993 [5]. Chicken PGCs can be stored at -196 C using a simple protocol, without losing their ability to transmit to the germline [6][7][8]. In addition, a novel method for long-term culture of PGCs that maintains their commitment to the germ cell lineage has recently been developed in chickens [9]. PGCs have therefore received ...

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Section: Discussioncontrasting

confidence: 85%

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confidence: 99%

X-irradiation Removes Endogenous Primordial Germ Cells (PGCs) and Increases Germline Transmission of Donor PGCs in Chimeric Chickens

et al. 2012

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“…The efficiency of germline chimera production was significantly improved by removal of blood from the recipient embryo (Naito et al, 1994a), or by application of Busulfan to the blastoderm (Nakamura et al, 2010) prior to PGC transfers. Furthermore, germline chimeras were successfully produced by transferring frozen/thawed PGCs into 2-day-old recipient embryos by Naito et al (1994b).…”

Section: Conservation Of Avian Genetic Resources Through Germline Chimentioning

confidence: 99%

Conservation of Avian Genetic Resources

Tajima

2013

J. Poult. Sci.

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Conservation of avian genetic resources is important, as 13.0% (1,313/10,064) of known avian species are currently categorized as endangered by the International Union for Conservation of Nature and Natural Resources (IUCN, 2012). Development of an integrated system for conservation of avian genetics is necessary; however, germplasm conservation systems developed for mammals cannot be applied directly to avians, primarily due to 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 into recipient embryos has enabled the conservation and retrieval of chicken genetic resources in their complete form. Furthermore, the possibility of propagating endangered wild birds through interspecies germline chimeras has been proposed.In the present review, the current status of avian genetic resource conservation is presented with a focus on the following 4 topics: 1) semen cryopreservation, 2) germline chimeras, 3) somatic nuclear transfer, and 4) other related technologies.

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“…Emulsification of busulfan and injection into chicken embryos was performed as described by Nakamura and colleagues [5], with minor modifications. A schematic diagram of busulfan emulsification and injection into eggs is shown in Figure 1.…”

Section: Methodsmentioning

confidence: 99%

“…After busulfan treatment, donor PGCs injected into the embryos migrated and colonized on the recipient gonads. The proportion of donor-derived offspring was also increased significantly [5,12]. However, little is known about the cellular responses of PGCs after busulfan treatment.…”

Section: Introductionmentioning

confidence: 99%

Compensatory proliferation of endogenous chicken primordial germ cells after elimination by busulfan treatment

et al. 2013

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IntroductionPrimordial germ cells (PGCs) are the major population of cells in the developing bilateral embryonic gonads. Little is known about the cellular responses of PGCs after treatment with toxic chemicals such as busulfan during embryo development. In this study, we investigated the elimination, restorative ability, and cell cycle status of endogenous chicken PGCs after busulfan treatment.MethodsBusulfan was emulsified in sesame oil by a dispersion-emulsifying system and injected into the chick blastoderm (embryonic stage X). Subsequently, we conducted flow cytometry analysis to evaluate changes in the PGC population and cell cycle status, and immunohistochemistry to examine the germ cell proliferation.ResultsResults of flow cytometry and immunohistochemistry analyses after busulfan treatment showed that the proportion of male PGCs at embryonic day 9 and female PGCs at embryonic day 7 were increased by approximately 60% when compared with embryonic day 5.5. This result suggests the existence of a compensatory mechanism in PGCs in response to the cytotoxic effects of busulfan. Results of cell cycling analysis showed that the germ cells in the G0/G1 phase were significantly decreased, while S/G2/M-phase germ cells were significantly increased in the treatment group compared with the untreated control group in both 9-day-old male and female embryos. In addition, in the proliferation analysis with 5-ethynyl-2′-deoxyuridine (EdU) incorporation, we found that the proportion of EdU-positive cells among VASA homolog-positive cells in the 9-day embryonic gonads of the busulfan-treated group was significantly higher than in the control group.ConclusionsWe conclude that PGCs enter a restoration pathway by promoting their cell cycle after experiencing a cytotoxic effect.

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

Cited by 63 publications

References 37 publications

X-irradiation Removes Endogenous Primordial Germ Cells (PGCs) and Increases Germline Transmission of Donor PGCs in Chimeric Chickens

X-irradiation Removes Endogenous Primordial Germ Cells (PGCs) and Increases Germline Transmission of Donor PGCs in Chimeric Chickens

Conservation of Avian Genetic Resources

Compensatory proliferation of endogenous chicken primordial germ cells after elimination by busulfan treatment

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