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Xie Y.,Northwest University, China | Xie Y.,Key Laboratory of Animal Biotechnology | Zhao X.,Northwest University, China | Zhao X.,Key Laboratory of Animal Biotechnology | And 4 more authors.
In Vitro Cellular and Developmental Biology - Animal | Year: 2013

Fetal fibroblast cells (FFCs) are often used as donor cells for somatic cell nuclear transfer (SCNT) because they are easy to culture and suitable for genetic manipulation. However, through genetic modification process, which required FFCs to be cultured in vitro for several passages, cells tended to age very rapidly and became inappropriate for SCNT. Human telomerase reverse transcriptase (hTERT) possessed the activity of human telomerase and maintains telomere in dividing cells; therefore, hTERT can be transfected into somatic cells to extend their lifespan. In this study, we transfected a Xinong Saanen Dairy Goat FFC line with hTERT. Then, we tested several characteristics of transfected cells, including growth curve, expression and activity of hTERT, tumorigenicity, and expression of oct4 and nanog. The result showed that hTERT could significantly extend the lifespan of transfected cells in vitro. hTERT mRNA was expressed in hTERT-transfected cells. Moreover, hTERT-transfected cells presented enhanced telomerase activity and longer telomere than untransfected cells at the same passage. On the other hand, hTERT-transfected cells can maintain normal karyotype even after several times of subculture in vitro. After inoculation of hTERT-transfected cells in nude mouse, none of them developed tumors on the vaccination site. Interestingly, transfection of hTERT can improve expression of nanog and oct4 in Xinong Saanen Dairy Goat FFCs, especially in low generation after transfection, but with increasing subculture, this effect gradually weakened. © 2012 The Society for In Vitro Biology. Source


Yu X.-L.,Northwest University, China | Yu X.-L.,Key Laboratory of Animal Biotechnology | Zhao X.-E.,Northwest University, China | Zhao X.-E.,Key Laboratory of Animal Biotechnology | And 4 more authors.
Journal of Integrative Agriculture | Year: 2015

The transcription factors, including OCT4, NANOG, and SOX2, played crucial roles in the maintenance of self-renewal and pluripotency in embryonic stem cells (ESCs). They expressed in preimplantation mammalian development with spatio-temporal pattern and took part in regulation of development. However, their expression and roles in goat had not been reported. In the present study, the expression of OCT4, NANOG, and SOX2 in goat preimplantation embryos both in vivo and in vitro were detected by real-time RCR and immunofluorescence. For in vivo fertilized embryos, the transcripts of OCT4, NANOG, and SOX2 could be detected from oocytes to blastocyst stage, their expression in morula and blastocyst stages was much higher than other stage. OCT4 protein was detected from oocyte to blastocyst, but the fluorescence was more located-intensive with nuclei from 8-cell stage, its expression present in both inner cell mass (ICM) and trophoblast cells (TE) at blastocyse stage. NANOG protein was similar to OCT4, the signaling of fluorescence completely focused on cell nuclei, while the SOX2 firstly showed nuclei location in morula. Comparing to in vivo fertilized embryo, the mRNA of these three transcription factors could be detected at 8-cell stage in parthenogenetic embryos (in vitro). Thereafter, the expressional level rose gradually along with embryo development. The locations of OCT4 and NANOG proteins were similar to in vivo fertilized embryos, and they located in cell nuclei from morula to blastocyst stage, while SOX2 protein firstly could be detected in cell nuclei at 8-cell stage. These differences suggested that OCT4, NANOG, and SOX2 played different function in regulating development of goat preimplantation embryos. These results may provide a novel insight to goat embryo development and be useful for goat ESCs isolation. © 2015, CAAS. Source


Wu H.,Key Laboratory of Animal Biotechnology | Wu Y.,Key Laboratory of Animal Biotechnology | Ai Z.,Key Laboratory of Animal Biotechnology | Ai Z.,Northwest University, China | And 8 more authors.
Stem Cells | Year: 2014

Vitamin C (Vc), also known as ascorbic acid, is involved in many important metabolic and physiological reactions in the body. Here, we report that Vc enhances the expression of Nanog and inhibits retinoic acid-induced differentiation of embryonic stem cells. We investigated Vc regulation of Nanog through Janus kinase/signal transducer and activator of transcription pathway using cell signaling pathway profiling systems, and further confirmed by specific pathway inhibition. Using overexpression and knockdown strategies, we demonstrated that STAT2 is a new positive regulator of Nanog and is activated by phosphorylation following Vc treatment. In addition, site mutation analysis identified that STAT2 physically occupies the Nanog promoter, which was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Taken together, our data suggest a role for Vc in Nanog regulation networks and reveal a novel role for STAT2 in regulating Nanog expression.© AlphaMed Press 2013. Source


Liu X.,Northwest Agriculture and Forestry University | Liu X.,Key Laboratory of Animal Biotechnology | Wei Q.,Northwest Agriculture and Forestry University | Wei Q.,Key Laboratory of Animal Biotechnology | And 8 more authors.
In Vitro Cellular and Developmental Biology - Animal | Year: 2015

Kunming mice are widely used in China; however, it is difficult to isolate embryonic stem cells (ESCs) in conventional derivation condition containing feeder cells and serum. 6-Bromoindirubin-3′-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, could facilitate the maintenance of pluripotency of ESCs. Therefore, BIO could be considered as a candidate to replace feeder cells and serum. On the other hand, in vitro fertilization (IVF) is an important technology in assisted reproduction. It is reported that there was some difference in gene expression between IVF and in vivo developed blastocyst. ESCs derived from IVF blastocyst could provide a valuable tool to research the effect of IVF on differentiation and development. In the present study, we established two novel ESC lines from IVF blastocyst of Kunming mice in a feeder- and serum-free condition containing 2.5 μM BIO. In this condition, expanded IVF blastocyst could spontaneously hatch from zonae pellucidae and attached to the gelatin-coated bottom of dishes. ESC-like outgrowth could be observed without overfull trophoblast cells. After further propagation, two Kunming mice ESC lines, designated as KMES1 and KMES2, were obtained. These two novel ESCs shared common morphological characteristics with other rodent ESCs, showed strong alkaline phosphatase activity, and expressed pluripotent markers, including Oct-4, Nanog, and SSEA-1. Embryoid body (EB) and teratoma test indicated that these ESCs could spontaneously differentiate into cells representative of all three embryonic germ layers. © 2015, The Society for In Vitro Biology. Source


Yang Z.,Northwest University, China | Yang Z.,Key Laboratory of Animal Biotechnology | Liu J.,Northwest University, China | Liu J.,Key Laboratory of Animal Biotechnology | And 14 more authors.
Cellular Reprogramming | Year: 2013

Novel stem cells expressing stage-specific embryonic antigen 3 (SSEA-3) reside among human dermal fibroblasts and are known as multilineage- differentiating stress-enduring (Muse) cells. They enhance the generation efficiency of induced pluripotent stem cells. However, Muse cells have only been found in humans. We aimed to isolate SSEA3-positive cells from terminally differentiated skin fibroblasts of adult goat and determine their pluripotency. Cell clusters from SSEA3+ populations possessed stem cell-like morphological features and normal karyotypes, were consistently positive for alkaline phosphatase, and expressed stem cell pluripotency markers. These SSEA3+ cells remained undifferentiated over eight passages in suspension culture and were able to differentiate into cells of all three germ layers in vitro and in vivo. Our combined findings suggest that a subset of adult stem cells expressing SSEA3 also exist among adult goat skin fibroblasts. We are the first to report that multipotent adult goat cells exist among terminally differentiated goat skin in suspension culture. Our results also provide a promising platform for generation of a transgenic goat, because the undifferentiated state of stem cells was thought to be more efficient as donor cells for somatic cell nuclear transfer. © 2013, Mary Ann Liebert, Inc. Source

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