Isolation and culture of homogeneous populations of primordial germ cells in chick
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Special cytoplasm, called germ plasm, that is essential for the differentiation of germ cells is localized in a particular region of Caenorhabditis elegans, Drosophila and Xenopus eggs. The mode of founder cell formation of germline, the origin and behavior of the germline granules, and the molecules localized in germline cells are compared in these organisms. The common characteristics of the organisms are mainly as follows. First, the founder cells of germline are established before the intiation of gastrulation. Second, the germline granules or their derivatives are always present in germline cells or germ cells throughout the life cycle in embryos, larvae, and adults. Lastly, among the proteins localized in the germ plasm, only Vasa protein or its homolog is detected in the germline cells or germ cells throughout the life cycle. As the protein of vasa homolog has been reported to be also localized in the germline‐specific structure or nuage in some of the organisms without the germ plasm, the possibility that the mechanism for differentiation of primordial germ cells is basically common in all organisms with or without the germ plasm is discussed.
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Primordial germ cells (PGCs) are the stem cells of the germline. Generally, germline induction occurs via zygotic factors or the inheritance of maternal determinants called germ plasm (GP). GP is packaged into ribonucleoprotein complexes within oocytes and later promotes the germline fate in embryos. Once PGCs are specified by either mechanism, GP components localize to perinuclear granular-like structures. Although components of zebrafish PGC germ granules have been studied, the maternal factors regulating their assembly and contribution to germ cell development are unknown. Here, we show that the scaffold protein Dazap2 binds to Bucky ball, an essential regulator of oocyte polarity and GP assembly, and colocalizes with the GP in oocytes and in PGCs. Mutational analysis revealed a requirement for maternal Dazap2 (MDazap2) in germ-granule maintenance. Through molecular epistasis analyses, we show that MDazap2 is epistatic to Tdrd7 and maintains germ granules in the embryonic germline by counteracting Dynein activity.
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Modification of the mouse germline traditionally has been achieved in two ways, introduction of DNA into a fertilized egg or into embryonic stem (ES) cells that can contribute to the germline of a chimeric mouse produced by injection of the altered ES cells into a host embryo. Primordial germ cells (PGCs) and oogonia from female embryos can be transferred into the ovarian sac of an adult animal into fertilizable oocytes that give rise to offspring. Following a brief description of germ cell development in the mouse, this chapter describes techniques for isolating and culturing PGCs. Advances in PGC isolation and culture techniques may provide the opportunity to manipulate these PGCs in vitro while retaining their ability to populate the germline. Initial studies suggest that introduction and expression of cloned DNA into Embryonic germ (EG) cell lines is likely to be more straightforward than into PGCs. EG cells may prove useful in overcoming some of the limitations encountered using ES cells.
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This paper reviews the origin , specification and the genes related to the origin , specification of primordial germ cells(PGCs) and sex differentiation, isolation and culture of PGCs and embryonic germ cells(EG) cells and its related problems.
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The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al., 2016.
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Abstract Plethodon cinereus is favorable for a study of the history of the germ line. The germ cells are large, while the gonads are small and show an almost diagrammatic structure. Germ cell degeneration is not extensive during ontogeny. As long as all the germ cells contain yolk during development, these cells are derived solely from germ cells. In Plethodon, yolk lasts in all germ celis through sex differentiation. A comparison of the number of mitoses necessary for the primordial germ cells to produce the numbers of germ cells in gonads where every germ cell contains yolk, with the mitoses necessary for the primordial cells to produce the adult complements of germ cells, shows that at least 72% of the increase of germ cells can be followed by yolk. A cytological study from the embryo through the adult sexual cycle gave no evidence that somiatic cells ever transform into germ cells. Mitoses are abundant in the testes of salamanders starved 4 months. This fact together with a statistical study on the adult male sexual cycle indicates that the germ cells present in the testis do not need to be augmented by transformed somatic cells. It is concluded that in Plethodon germ cells alone give rise to germ cells, and that the germ line is continuous.
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Abstract Germline chimeric chickens were produced by transfer of primordial germ cells from White Leghorn to Barred Plymouth Rock, and vice versa. Blood was collected from stage 13–15 embryos and primordial germ cells were concentrated by Ficoll density gradient centrifugation. Approximately 200 primordial germ cells were injected into the bloodstream through the dorsal aorta of stage 14–15 recipient embryos from which blood had been drawn via the dorsal aorta prior to the injection. Intact embryos were also prepared as recipients for White Leghorns only. The manipulated embryos were cultured in recipient eggshells until hatching. Germline chimerism of the chickens reaching maturity was examined by mating them with Barred Plymouth Rocks and donor‐derived offspring were identified based on their feather color. The efficiency of production of germline chimeras was 95% (19/20). When primordial germ cells were transferred from White Leghorn to Barred Plymouth Rock, the average frequency of donor‐derived offspring was 81% for three male chimeras (96% for one female chimera), and it was ∼3.5 times higher for transfer in the opposite direction (23% for 6 male chimeras). Removing blood from recipient embryos prior to primordial germ cell injection enhanced the frequency of donor‐derived offspring by 10% in resulting male chimeras. Male chimeras produced donor‐derived offspring more frequently (∼3.8 times) than female chimeras. Increases, decreases, or no changes were observed in the frequency of donor‐derived offspring from the germline chimeras with increasing age. Male to female ratio of the offspring derived from the donor primordial germ cells did not deviate significantly in male and female chimeras, suggesting that primordial germ cells that have different sex from recipient embryos could not differentiate into functional gametes. The technique for primordial germ cell transfer employed in this experiment is simple to perform and resulted in the efficient production of germline chimeras with high transmission rates of donor‐derived gametes. This system provides a powerful tool for avian embryo manipulation. © 1994 Wiley‐Liss, Inc.
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