Migration of primordial germ cells isolated from embryonic blood into the gonads after transfer to stage X blastoderms and detection of germline chimaerism by PCR

Naito, Mitsuru; Sano, Akiko; Harumi, Takashi; Matsubara, Yuko; Kuwana, Takashi

doi:10.1080/00071660400014325

Cited by 28 publications

(14 citation statements)

References 43 publications

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“…Freshly collected PGCs usually migrate to the recipient gonads by transferring them into the stage X blastoderm (Naito et al, 2004) or bloodstream (Yasuda et al, 1992;Tajima et al, 1993;Naito et al, 1994aNaito et al, , 1994bNaito et al, , 2010. On the other hand, when cultured PGCs were transferred to the coelomic epithelium of recipient embryos, they were able to enter the gonads, as in our previous report (Naito et al, 2011).…”

Section: Discussionmentioning

confidence: 71%

Expression of GFP Gene in Cultured PGCs Isolated from Embryonic Blood and Incorporation into Gonads of Recipient Embryos

2012

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The present study was conducted to develop a technique for generating primordial germ cells expressing GFP gene and introducing them into gonads of recipient embryos. Primordial germ cells isolated from embryonic blood were cultured on feeder cells for more than 40 days. They proliferated, occasionally formed cell colonies, and showed the characteristics of germline cells as detected by anti-CVH antibody. The cultured PGCs were transferred to the stage X blastoderm, bloodstream of stages 14-15 embryos, and the coelomic epithelium of stages 17-19 embryos, and examined to determine whether they could migrate to the gonads of recipient embryos. As a result, they successfully entered the gonads of recipient embryos when they were transferred to the coelomic epithelium, although they failed to migrate to the gonads of recipient embryos when they were transferred to the stage X blastoderm or bloodstream. The cultured PGCs were then transfected with GFP gene by nucleofection and selected for PGCs expressing GFP gene in the presence of G418. Cultured PGCs expressing GFP gene proliferated slowly, forming cell colonies, and successfully entering the gonads by transferring into the coelomic epithelium of recipient embryos. Those results suggest that gene transfer into the chicken germline is possible via cultured PGCs, and that the PGC culture system thus holds enormous possibilities for avian embryo manipulation.

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Section: Discussionmentioning

confidence: 71%

Expression of GFP Gene in Cultured PGCs Isolated from Embryonic Blood and Incorporation into Gonads of Recipient Embryos

2012

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“…Also, as mentioned above, the possibility exists of discovering freemartins with either hematopoietic cells alone as reported [41] or of gonads alone, which can be suggested from the studies in chickens [57].…”

Section: Freemartinsmentioning

confidence: 94%

Chimerism and tetragametic chimerism in humans: implications in autoimmunity, allorecognition and tolerance

et al. 2007

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The presence of cells or tissues from two individuals, chimeras, or the presence of cells and tissues that include the gonads, tetragametic chimerism can be detected by the analysis of cytogenetics and analysis of polymorphic genetic markers, using patterns of pedigree inheritance. These methodologies include determination of sex chromosomes, major histocompatibility complex (MHC) polymorphisms and panels of short tandem repeats (STRs) that include mitochondrial DNA markers. Studies routinely involve cases of temporal chimerism in blood transfusion, or following allotransplantation to measure the outcome of the organ, lymphopoietic tissues or bone marrow grafts. Demonstration of persistent chimerism is usually discovered in cases of inter-sexuality due to fusion of fraternal twins or in cases of fusion of embryos with demonstrable allogeneic monoclonality of blood which, excluded maternity or paternity when blood alone is used as the source of DNA. In single pregnancies it is possible to produce two kinds of microchimerism: feto-maternal and materno-fetal, but in cases of fraternal twin pregnancies it is possible to identify three different kinds which are related to cases of vanishing twins that can be identified during pregnancy by imaging procedures; (1) hematopoietic, (2) gonadal, and (3) freemartins when the twins have different sex and the individual born is a female with either gonadal or both gonadal and hematopoietic tissues. Fraternal twin pregnancies can also produce fusion of embryos. Such cases could be of different sex presenting with inter-sexuality or in same sex twins. One of such cases, the best studied, showed evidence of chimerism and tetragametism. In this regard, the case was studied because of disputed maternity of two of her three children. All tissues studied, except for the blood, demonstrated four genetic components but only two in her blood of four possible showed allogeneic monoclonality consistent with the interpretation that her blood originated from one hematopoietic stem cell. Also, microchimerism, due to traffic of cells via materno-fetal or feto-maternal has been prompted by reports of their potential association with the development of autoimmune disorders including systemic lupus erythematosus (SLE) and systemic sclerosis, and in allotransplantation. In addition, their relevance of chimerism in the positive and negative selection of T cells in the thymus has not been addressed. T lymphocytes play a central role in controlling the acquired immune response and furthermore serve as crucial effector cells through antigen specific cytotoxic activity and the production of soluble mediators. Central tolerance is established by the repertoire selection of immature T lymphocytes in the thymus, avoiding the generation of autoreactive T cells. Expression of chimeric antigens in the thymus could modify the generation of specific T cell clones in chimeric subjects and these mechanisms could be important in the induction of central tolerance against foreign antigens important in allo-transp...

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“…Recent reports indicated that PGCs remain restricted to the germ line [13,14]. Chicken EG and ES cells contribute substantially to somatic tissues but not to the germ line [15].…”

Section: Isolation and Culture Of Chicken Pgcsmentioning

confidence: 99%

Isolation, Culture and Biological Characteristics of Primordial Germ Cells from Beijing Fatty Chicken

Wang

Hou

et al. 2010

Journal of Reproduction and Development

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Abstract. The aim of this study was to explore the isolation and culture process of Beijing fatty chicken primordial germ cells (PGCs) and investigate their biological characteristics. The PGCs isolated from the genital ridges of Beijing fatty chicken (Gallus domesticus) embryos after 5.5 days of incubation were co-cultured with mice embryonic fibroblasts (MEF). The results showed that the PGCs of the Beijing fatty chicken were positive for periodic acid Schiff (PAS) and alkaline phosphatase (AKP) staining. These cells could proliferate for a prolonged time in vitro and maintain diploid karyotype. Immunocytochemical staining showed that they expressed SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81, and real-time PCR showed that they expressed Cvh, CDH and Dazl. They could form simple embryoid bodies and differentiate into osteoblasts in vitro. In addition, after transfected with pEGFP-N3, pEYFP-N1 and pDsRed-N1 vectors by liposomal transfection, enhanced green, yellow and red fluorescent protein-positive cells could be visualized using a laser confocal microscope. The above results suggested that PGCs from the Beijing fatty chicken not only had strong self-renewal ability, but also had the potential to differentiate towards mesoblast cells. These cells are suitable for genetic manipulation as nuclear donors. s progenitors of spermatogonia and oogonia, PGCs are characterized by their pluripotency, and therefore represent a good model for the study of embryo development in vitro. Owing to their physiological and developmental characteristics, the avian, especially the chicken, has great value in transgenic research. An efficient transfer system of using chicken gonadal PGCs for producing germline chimeras has been established [1]. Production of transgenic chickens will supply tremendous bioactive materials for human pharmaceutics and functional food development. In chicken germ cell research, it is occasionally considered that SSEA-1 and PAS-positive cells are presumptive PGCs, and the pluripotency of candidate PGCs has been confirmed by induction of germline transmission via the transfer of cells into recipient embryos [2]. Apparently, the former method is somewhat insufficient to fully characterize chicken PGCs, while the latter is too complicated and time-consuming. Therefore, additional effort to identify the characterization of chicken PGCs comprehensively is urgently required for further development of avian transgenic systems.In this report, PGCs were isolated from the genital ridges of Beijing fatty chicken embryos. After proliferating in vitro for a prolonged period, they can maintain diploid karyotype, express a set of typical markers of ES cells and differentiate into osteoblasts in vitro. Furthermore, the results demonstrated that the chicken PGCs had a relatively high cloning efficiency and could be used as nuclear donors to accept transgenes readily. Materials and Methods Preparation of mouse embryonic fibroblasts (MEF)Fibroblasts were isolated from mouse embryos on day 13.5, and cultured in Dulbecco's modifi...

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Migration of primordial germ cells isolated from embryonic blood into the gonads after transfer to stage X blastoderms and detection of germline chimaerism by PCR

Cited by 28 publications

References 43 publications

Expression of GFP Gene in Cultured PGCs Isolated from Embryonic Blood and Incorporation into Gonads of Recipient Embryos

Expression of GFP Gene in Cultured PGCs Isolated from Embryonic Blood and Incorporation into Gonads of Recipient Embryos

Chimerism and tetragametic chimerism in humans: implications in autoimmunity, allorecognition and tolerance

Isolation, Culture and Biological Characteristics of Primordial Germ Cells from Beijing Fatty Chicken

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