Restriction of proliferation of primordial germ cells by the irradiation of Japanese quail embryos with soft X-rays

Li, Haichang; Kono, Hiroshi; Matsui, Kanji; Ono, Tamao

doi:10.1016/s1095-6433(01)00375-0

Cited by 35 publications

(16 citation statements)

References 34 publications

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“…6, A'''-D'''; green) could be distinguished in the gonads by the fluorescence of the donor and recipient PGCs. Recipient PGCs were mainly detected in the chimeric gonads, since the endogenous PGCs of the recipient embryo [21][22][23] were not removed (Fig. 6, A'''-D''').…”

Section: Discussionmentioning

confidence: 99%

A Novel Method to Isolate Primordial Germ Cells and Its Use for the Generation of Germline Chimeras in Chicken1

Yamamoto

Usui

Nakamura

et al. 2007

Biology of Reproduction

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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 germlinespecific 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. developmental biology, early development, gamete biology, gene regulation

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Section: Discussionmentioning

confidence: 99%

A Novel Method to Isolate Primordial Germ Cells and Its Use for the Generation of Germline Chimeras in Chicken1

Yamamoto

Usui

Nakamura

et al. 2007

Biology of Reproduction

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“…To determine whether the V-ATPase is normally upregulated in existing cells or produced by a new cell population generated in response to amputation, we irradiated larvae, a procedure that abolishes cell proliferation (Li et al, 2001;Salo and Baguna, 1985). Irradiated larvae still upregulated V-ATPase expression in the wound (Fig.…”

Section: Cell-surface Expression Of V-atpase Is Rapidly Induced In Exmentioning

confidence: 99%

H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induceXenopustail regeneration

Adams¹,

Masi²,

Levin³

2007

Development

253

311

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In many systems, ion flows and long-term endogenous voltage gradients regulate patterning events, but molecular details remain mysterious. To establish a mechanistic link between biophysical events and regeneration, we investigated the role of ion transport during Xenopus tail regeneration. We show that activity of the V-ATPase H+ pump is required for regeneration but not wound healing or tail development. The V-ATPase is specifically upregulated in existing wound cells by 6 hours post-amputation. Pharmacological or molecular genetic loss of V-ATPase function and the consequent strong depolarization abrogates regeneration without inducing apoptosis. Uncut tails are normally mostly polarized, with discrete populations of depolarized cells throughout. After amputation, the normal regeneration bud is depolarized, but by 24 hours post-amputation becomes rapidly repolarized by the activity of the V-ATPase, and an island of depolarized cells appears just anterior to the regeneration bud. Tail buds in a non-regenerative `refractory' state instead remain highly depolarized relative to uncut or regenerating tails. Depolarization caused by V-ATPase loss-of-function results in a drastic reduction of cell proliferation in the bud, a profound mispatterning of neural components, and a failure to regenerate. Crucially, induction of H+ flux is sufficient to rescue axonal patterning and tail outgrowth in otherwise non-regenerative conditions. These data provide the first detailed mechanistic synthesis of bioelectrical,molecular and cell-biological events underlying the regeneration of a complex vertebrate structure that includes spinal cord, and suggest a model of the biophysical and molecular steps underlying tail regeneration. Control of H+ flows represents a very important new modality that, together with traditional biochemical approaches, may eventually allow augmentation of regeneration for therapeutic applications.

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“…However, drawing blood from recipient embryos prior to the transfer of donor PGCs resulted in only a slight improvement in germline chimerism because only about a third of the total blood was removed, leaving endogenous PGCs intact [18]. Irradiation with soft (low-energy) X-rays can effectively reduce the number of endogenous PGCs in chickens [19][20][21] and quail [22]. Because the sterilizing effects on PGCs are restricted to the irradiation period, donor PGCs would proliferate in irradiated recipient embryos after transplantation.…”

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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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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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Restriction of proliferation of primordial germ cells by the irradiation of Japanese quail embryos with soft X-rays

Cited by 35 publications

References 34 publications

A Novel Method to Isolate Primordial Germ Cells and Its Use for the Generation of Germline Chimeras in Chicken1

A Novel Method to Isolate Primordial Germ Cells and Its Use for the Generation of Germline Chimeras in Chicken1

H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induceXenopustail regeneration

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

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