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 In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Intekhab Islam ◽  
Gopu Sriram ◽  
Mingming Li ◽  
Yu Zou ◽  
Lulu Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Green Fluorescent Protein ◽  
Bone Regeneration ◽  
Osteogenic Differentiation ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Differentiation Potential ◽  
Green Fluorescent

Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs) are potentially an unlimited source of healthy and functional osteoprogenitors (OPs) that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs) expressing green fluorescent protein (GFP) and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.

scholarly journals Embryonic Stem Cells and Transgenic Mice Ubiquitously Expressing a Tau-Tagged Green Fluorescent Protein

2000 ◽  
Vol 228 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Thomas Pratt ◽  
Linda Sharp ◽  
Jenny Nichols ◽  
David J. Price ◽  
John O. Mason
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Embryonic Stem Cells ◽  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Green Fluorescent

scholarly journals In Vivo and In Vitro Tissue-Specific Expression of Green Fluorescent Protein Using the Cre-Lox System in Mouse Embryonic Stem Cells

Stem Cells ◽  
2005 ◽  
Vol 23 (1) ◽  
pp. 10-15 ◽  
Author(s):  
Jan Schindehütte ◽  
Hidefumi Fukumitsu ◽  
Patrick Collombat ◽  
Gundula Griesel ◽  
Christopher Brink ◽  
...  
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Embryonic Stem Cells ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Specific Expression ◽  
Tissue Specific Expression ◽  
Green Fluorescent

Generation of Green Fluorescent Protein-Expressing Monkey Embryonic Stem Cells

2006 ◽  
pp. 305-312
Author(s):  
Yutaka Suzuki ◽  
Tatsuyuki Takada ◽  
Nae Kadota ◽  
Yasushi Kondo ◽  
Ryuzo Torii
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Embryonic Stem Cells ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Green Fluorescent

Maintenance of Pluripotency in Human Embryonic Stem Cells Stably Over-expressing Enhanced Green Fluorescent Protein

2004 ◽  
Vol 13 (6) ◽  
pp. 636-645 ◽  
Author(s):  
Yi-Ping Liu ◽  
Oksana V. Dovzhenko ◽  
Mark A. Garthwaite ◽  
Svetlana V. Dambaeva ◽  
Maureen Durning ◽  
...  
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Green Fluorescent

128 MULTIPLICATION OF 8-CELL EMBRYOS BY AGGREGATION OF A SINGLE ENHANCED GREEN FLUORESCENT PROTEIN-LABELED BLASTOMERE WITH PUTATIVE TETRAPLOID EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 168
Author(s):  
M. I. Hiriart ◽  
R. J. Bevacqua ◽  
R. Fernandez-Martin ◽  
D. F. Salamone
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Egfp Expression ◽  
Simple Method ◽  
Transgenic Embryos ◽  
Green Fluorescent

Isolated blastomeres from 2- and 4-cell embryos are able to generate live offspring. However, the development of each cell of an 8-cell embryo is limited. Tetraploid embryos are used for aggregation with other embryos, embryonic stem cells, and iPS cells, and they are selected against during development of the fetal tissues, but persist in extraembryonic membranes. The objective of this work was to generate a new and simple method for cloning 8-cell bovine embryos and also to explore more efficient methods to multiply transgenic embryos by aggregation of each blastomere from a day-3 embryo with putative tetraploid embryos. To this aim, bovine cumulus–oocyte complexes were in vitro matured in standard conditions and subjected to IVF (day 0) according to Bracket and Oliphant (1975). After IVF, a group of presumptive zygotes was injected with ooplasmic vesicles incubated with 50 ng mL–1 of linearized pCX–egfp. Other group was cultured for 25 additional hours (day 1). At that time 2-cell embryos were electrofused twice at 40V for 25 μs at 100-ms intervals to generate putative tetraploid embryos, visualised as a single blastomere 1 h after the fusion pulse (fused embryos, F). Two aggregation groups were included. A synchronic group (S): IVF for the production of both transgenic embryos and fused embryos was done on the same day; and an asynchronic group (AS): IVF for transgenic embryos took place 1 day before IVF for fused embryos production, so embryos from the A group were younger. Controls consisted of the same S and AS groups, but no fusion was included (NF). On day 3, the enhanced green fluorescent protein [EGFP(+)] blastomeres were selected. Using the well of well system, 1 or 2 embryos of each fusion group (S or AS and F or NF) were removed of their ZP and aggregated in a microwell with one EGFP(+) blastomere from a 5- to 8-cell stage embryo (day 3). In vitro development of the aggregates and green fluorescent protein expression localization of blastocysts were analysed. Blastocysts were obtained for all groups; however, the 2A-F and 2A-NF groups showed the highest rates (44%, P < 0.05) compared with one embryo aggregation. The highest aggregation rates of the EGFP(+) blastomere were observed for 2A-F (67%) and 2A-NF (44%) groups, too. A very poor integration was noted in the 2S-NF (100%), 2S-F (94%), 1A-NF (89%), and 1S-NF (80%) groups. Localised EGFP distribution was also high in the 2A-F group (42%). In all cases, EGFP expression seemed to localise by the inner cell mass. We demonstrated that it is possible to multiply 8-cell embryos of genetic value and also transgenic embryos, in theory reducing mosaicism rates in future offspring. Moreover, our results give rise to the possibility of using EGFP like a reporter gene that could be used to evaluate aggregation efficiency by a fluorescence microscope.

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