Cryopreservation of green fluorescent protein (GFP)-labeled primordial germ cells with GFP fused to the 3′ untranslated region of the nanos gene by vitrification of Japanese eel (Anguilla japonica) somite stage embryos1

2012 ◽  
Vol 90 (12) ◽  
pp. 4256-4265 ◽  
Author(s):  
Y. Kawakami ◽  
M. Ishihara ◽  
T. Saito ◽  
T. Fujimoto ◽  
S. Adachi ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Germ Cells ◽  
Untranslated Region ◽  
Fluorescent Protein ◽  
Primordial Germ Cells ◽  
Anguilla Japonica ◽  
Japanese Eel ◽  
Somite Stage ◽  
Green Fluorescent

scholarly journals Production of fertile zebrafish (Danio rerio) possessing germ cells (gametes) originated from primordial germ cells recovered from vitrified embryos

Reproduction ◽  
2010 ◽  
Vol 139 (4) ◽  
pp. 733-740 ◽  
Author(s):  
Shogo Higaki ◽  
Yoshiki Eto ◽  
Yutaka Kawakami ◽  
Etsuro Yamaha ◽  
Noriko Kagawa ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Ethylene Glycol ◽  
Germ Cells ◽  
Danio Rerio ◽  
Recovery Rate ◽  
Fluorescent Protein ◽  
Primordial Germ Cells ◽  
Ice Formation ◽  
Mature Fish ◽  
Green Fluorescent

This study aimed to produce fertile zebrafish (Danio rerio) possessing germ cells (gametes) that originated from cryopreserved primordial germ cells (PGCs). First, to improve the vitrification procedure of PGCs in segmentation stage embryos, dechorionated yolk-intact and yolk-removed embryos, the PGCs of which were labeled with green fluorescent protein, were cooled rapidly after serial exposures to equilibration solution (ES) and vitrification solution (VS), which contained ethylene glycol, DMSO, and sucrose. Yolk removal well prevented ice formation in the embryos during cooling and improved the viability of cryopreserved PGCs. The maximum recovery rate of live PGCs in the yolk-removed embryos vitrified after optimum exposure to ES and VS was estimated to be about 90%, and about 50% of the live PGCs showed pseudopodial movement. Next, to elucidate the ability of cryopreserved PGCs to differentiate into functional gametes, PGCs recovered from the yolk-removed embryos (striped-type) that were vitrified under the optimum exposure to ES and VS were transplanted individually into 218 sterilized recipient blastulae (golden-type). Two days after the transplantation, 7.5% (14/187) of morphologically normal embryos had PGC(s) in the genital ridges. Six (5 males and 1 female) of the 14 recipient embryos developed into mature fish and generated progeny with characteristics inherited from PGC donors. In conclusion, we demonstrated the successful cryopreservation of PGCs by vitrification of yolk-removed embryos and the production of fertile zebrafish possessing germ cells that originated from the PGCs in vitrified embryos.

scholarly journals Visualization and motility of primordial germ cells using green fluorescent protein fused to 3'UTR of common carp nanos-related gene

Aquaculture ◽  
2011 ◽  
Vol 317 (1-4) ◽  
pp. 245-250 ◽  
Author(s):  
Yutaka Kawakami ◽  
Taiju Saito ◽  
Takafumi Fujimoto ◽  
Rie Goto-Kazeto ◽  
Eisuke Takahashi ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Common Carp ◽  
Germ Cells ◽  
Fluorescent Protein ◽  
Primordial Germ Cells ◽  
Related Gene ◽  
Green Fluorescent

250 ALL REGIONS OF THE MOUSE EPIDIDYMIS ARE ABLE TO PHAGOCYTIZE IMMATURE SPERMATOGENIC CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 272
Author(s):  
P. Ramos-Ibeas ◽  
E. Pericuesta ◽  
R. Fernandez-Gonzalez ◽  
M. A. Ramirez ◽  
A. Gutierrez-Adan
Keyword(s):  
Quality Control ◽  
Green Fluorescent Protein ◽  
Germ Cells ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Sperm Quality ◽  
Seminiferous Tubules ◽  
Spermatogenic Cells ◽  
Green Fluorescent ◽  
Immature Cells

Successful mammalian fertilization requires gametes with an intact structure and functionality. Although it is well known that epididymal functions are sperm maturation, sustenance, transport, and storage, there is controversial information about its role in sperm quality control, and it has been suggested that some regions of the rat epididymis are able to phagocytize germ cells. Our objective was to analyse whether different segments of the mouse epididymal epithelium act as a selection barrier for abnormal spermatogenic cells by removing immature cells from the lumen by phagocytosis. To detect the presence of immature germ cells along the epididymis, transgenic mice expressing enhanced green fluorescent protein under a Deleted in Azoospermia-Like (mDazl) promoter were generated. The transgenic animals express specifically enhanced green fluorescent protein in spermatogonias, spermatocytes, and spermatids; thus, immature spermatogenic cells can be easily identified by fluorescence microscopy. Colchicine, a microtubule disruptor that leads to severe alterations in the architecture of the seminiferous tubules, was administered in the rete testis to induce the release of immature germ cells into the epididymis. Mice were killed daily, from Day 1 to 8 post-administration, and epididymides were collected and observed under a fluorescence stereoscope to determine the transit of immature germ cells along the epididymis. Epididymides from control mice without colchicine administration were also collected. Fluorescent immature germ cells were present in the caput epididymis 24 h after colchicine administration, and they progressed through the corpus and cauda, leaving the epididymis 7 days after colchicine administration. After fluorescence observation, epididymides were fixed, sectioned, and stained with hematoxylin solution. Immature germ cells and phagosomes were not observed in control epididymides. By contrast, the presence of phagosomes in the principal cells of the epididymal epithelium containing immature germ cells in different degrees of degradation was observed by light microscopy in mice injected with colchicine. Phagocytosis was observed along the epididymis following the main wave of fluorescent immature cells. Thus, when immature cells had reached the corpus epididymis, phagocytosis was detected in several segments of the caput epididymis. Later, once the immature cells had arrived to the cauda epididymis or had abandoned the epididymis, phagocytosis was observed in the corpus and cauda epididymis. The presence of phagosomes was observed in all epididymal tubules within a phagocytosis area. In conclusion, we demonstrated that the epididymal epithelium is engaged in sperm quality control by clearing immature germ cells after a massive shedding into the epididymal lumen, and that this phenomenon is not restricted to a specific segment of the epididymis.

Characterisation of the deleted in azoospermia like (Dazl)–green fluorescent protein mouse model generated by a two-step embryonic stem cell-based strategy to identify pluripotent and germ cells

2016 ◽  
Vol 28 (11) ◽  
pp. 1741 ◽  
Author(s):  
Priscila Ramos-Ibeas ◽  
Eva Pericuesta ◽  
Raúl Fernández-González ◽  
Alfonso Gutiérrez-Adán ◽  
Miguel Ángel Ramírez
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Transgenic Mice ◽  
Germ Cells ◽  
Fluorescent Protein ◽  
Preimplantation Embryo ◽  
Embryonic Stem ◽  
Deleted In Azoospermia ◽  
Green Fluorescent

The deleted in azoospermia like (Dazl) gene is preferentially expressed in germ cells; however, recent studies indicate that it may have pluripotency-related functions. We generated Dazl–green fluorescent protein (GFP) transgenic mice and assayed the ability of Dazl-driven GFP to mark preimplantation embryo development, fetal, neonatal and adult tissues, and in vitro differentiation from embryonic stem cells (ESCs) to embryoid bodies (EBs) and to primordial germ cell (PGC)-like cells. The Dazl-GFP mice were generated by a two-step ESC-based strategy, which enabled primary and secondary screening of stably transfected clones before embryo injection. During preimplantation embryo stages, GFP was detected from the zygote to blastocyst stage. At Embryonic Day (E) 12.5, GFP was expressed in gonadal ridges and in neonatal gonads of both sexes. In adult mice, GFP expression was found during spermatogenesis from spermatogonia to elongating spermatids and in the cytoplasm of oocytes. However, GFP mRNA was also detected in other tissues harbouring multipotent cells, such as the intestine and bone marrow. Fluorescence was maintained along in vitro Dazl-GFP ESC differentiation to EBs, and in PGC-like cells. In addition to its largely known function in germ cell development, Dazl could have an additional role in pluripotency, supporting these transgenic mice as a valuable tool for the prospective identification of stem cells from several tissues.

scholarly journals Genetic constructs creation using Golden Gate cloning method

2019 ◽  
Vol 25 ◽  
pp. 190-196 ◽  
Author(s):  
O. I. Varchenko ◽  
B. M. Krasyuk ◽  
A. A. Fedchunov ◽  
O. V. Zimina ◽  
M. F. Parii ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Untranslated Region ◽  
Fluorescent Protein ◽  
Regulatory Elements ◽  
Golden Gate ◽  
Genes Encoding ◽  
Green Fluorescent ◽  
Genetic Constructs

Aim. Creation of genetic constructions to study the effects of various regulatory elements, namely promoters, on the expression of GFP reporter protein. Methods. For creation genetic constructs, the method of molecular cloning Golden Gate was used, which allows the rapid creation of genetic vectors using IIS type restriction enzymes and T4 DNA liga-ses. Results. For research six different promoters were selected, namely the 35S CaMV (Cauliflower Mosaic Virus), double 35S CaMV promoter, promoters of the RbcS2B and RbcS1B genes encoding a small subunit of ribulozobisphosphate carboxylase (RuBisCo) isolated from Arabidopsis thaliana (L.) Heynh.; promoters of genes encoding chlorophyll a-b binding proteins (LHB1B1 and LHB1B2) also isolated from A. thaliana (L.) Heynh. All transcription units additionally contained the following elements: the 5'-untranslated region Ω sequence (5’UTR Ω) from the tobacco mosaic virus TMV (Tobacco Mosaic Virus); the coding sequence of the gene gfp (Green Fluorescent Protein) isolated from A. victoria and the 35S Terminator CaMV with the polyadenylation signal and the 3'-untranslated region sequence. As a result, six genetic constructs with different regulatory elements, namely promoters, have been created. Conclusions. To study the effects of various regulatory elements, namely promoters, on the expression of a GFP repor-ter protein in transient or stable genetic transformation of plants the created genetic constructs can be used.Keywords: cloning, genetic constructs, promoters, Green Fluorescent Protein (GFP).

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