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Find video protocols related to scientific articles indexed in Pubmed.
Generation of mastitis resistance in cows by targeting human lysozyme gene to ?-casein locus using zinc-finger nucleases.
Proc. Biol. Sci.
PUBLISHED: 01-01-2014
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Mastitis costs the dairy industry billions of dollars annually and is the most consequential disease of dairy cattle. Transgenic cows secreting an antimicrobial peptide demonstrated resistance to mastitis. The combination of somatic cell gene targeting and nuclear transfer provides a powerful method to produce transgenic animals. Recent studies found that a precisely placed double-strand break induced by engineered zinc-finger nucleases (ZFNs) stimulated the integration of exogenous DNA stretches into a pre-determined genomic location, resulting in high-efficiency site-specific gene addition. Here, we used ZFNs to target human lysozyme (hLYZ) gene to bovine ?-casein locus, resulting in hLYZ knock-in of approximately 1% of ZFN-treated bovine fetal fibroblasts (BFFs). Gene-targeted fibroblast cell clones were screened by junction PCR amplification and Southern blot analysis. Gene-targeted BFFs were used in somatic cell nuclear transfer. In vitro assays demonstrated that the milk secreted by transgenic cows had the ability to kill Staphylococcus aureus. We report the production of cloned cows carrying human lysozyme gene knock-in ?-casein locus using ZFNs. Our findings open a unique avenue for the creation of transgenic cows from genetic engineering by providing a viable tool for enhancing resistance to disease and improving the health and welfare of livestock.
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Gender depended potentiality of differentiation of human umbilical cord mesenchymal stem cells into oocyte-Like cells in vitro.
Cell Biochem. Funct.
PUBLISHED: 01-28-2013
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Recent studies have demonstrated that germ-like cells could be differentiated from human umbilical cord mesenchymal stem cells (hUC-MSCs) in vitro. Whether the sexuality of hUC-MSCs affects the formation efficiency of germ-like cells derived from hUC-MSCs is still unclear. To clearly test the formation efficiency of oocyte-like cells from male and female hUC-MSCs, obtained hUC-MSCs were induced by 20% follicular fluid (FF) according to the method that has been proved by our previous studies. Results showed that hUC-MSCs differentiated into oocyte-like structures and expressed germ cell makers. It was noted that the presence of advanced oocyte-like cells in male hUC-MSCs (m-hUC-MSCs) was similar as that in female hUC-MSCs (f-hUC-MSCs); however, the expression of germ cells specific markers in m-hUC-MSCs was delayed compared with that in f-hUC-MSCs. In addition, immunofluorescence analysis demonstrated that germ cell-specific markers, Oct4, Vasa, Dazl, ZP2, ZP3 and Stra8, were expressed on the 14th day after induction in both f-hUC-MSCs and m-hUC-MSCs. However, the size of oocyte-like cells from f-hUC-MSCs was larger than that in m-hUC-MSCs. The level of secreted oestradiol was significantly higher in f-hUC-MSCs than m-hUC-MSCs. We sought to determine whether critical germ cells transcription factor-Figl? will promote the development of oocyte-like cells. Some germ cell-specific markers were increased when exogenous Figl? was transfected into hUC-MSCs. This process implied that germ-like cells might be produced by over-expression of exogenous germ cell-specific gene, and this process was similar as that in production of germ cells in induced pluripotent stem cells (iPSCs). Finally, to verify the feasibility that hUC-MSCs differentiate into germ cells, hUC-MSCs were transplanted into seminiferous tubules and kidney capsule of mouse, respectively, and we found the transplanted cells differentiated into germ-like cells in recipients seminiferous tubules and kidney capsule. This study will provide a simple model to study mammalian germ cell specification using hUC-MSCs in vitro.
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Pluripotent male germline stem cells from goat fetal testis and their survival in mouse testis.
Cell Reprogram
PUBLISHED: 04-09-2011
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Male germline stem cells (mGSCs) are stem cells present in male testis responsible for spermatogenesis during their whole life. Studies have shown that mGSCs can be derived in vitro and resemble embryonic stem cells (ESCs) properties both in the mouse and humans. However, little is know about these cells in domestic animals. Here we report the first successful establishment of goat GSCs derived from 2-5-month fetal testis, and developmental potential assay of these cells both in vitro and in vivo. These cells express pluripotent markers such as Oct4, Sox2, C-myc, and Tert when cultured as human ESCs conditions. Embryoid bodies (EBs) formed by goat mGSCs were induced with 2?×?10(-6) M retinoic acid (RA). Immunofluorescence analysis showed that some cells inside of the EBs were positive for meiosis marker-SCP3, STRA8, and germ cell marker-VASA, and haploid marker-FE-J1, PRM1, indicating their germ cell lineage differentiation. Some cells become elongated sperm-like cells after induction. Approximately 34.88% (30/86) embryos showed cleavage and four embryos were cultured on murine fibroblast feeder and formed small embryonic stem like colonies. However, most stalled at four-cell stage after intracytoplasmic sperm injection (ICSI) of these cells. Transplantation of DAPI labeled mGSCs into the seminiferous tubules of busulfan-treated mice, and showed that mGSCs can colonize, self-renew, and differentiate into germ cells. Thus, we have established a goat GSC cell line and these cells could be differentiated into sperm-like cells in vivo and sperms in vitro, providing a promising platform for generation of transgenic goat for production of specific humanized proteins.
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Retinol (vitamin A) maintains self-renewal of pluripotent male germline stem cells (mGSCs) from adult mouse testis.
J. Cell. Biochem.
PUBLISHED: 02-11-2011
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Studies have shown that male germline stem cells (mGSCs), which are responsible for maintaining spermatogenesis in the male, could be obtained from mouse and human testis. However, the traditional cultural methods were mostly dependent on serum and feeder, and the initial mGSCs were either obtained from neonatal mice or the detailed description of its potency and origin was not provided. Here we reported a novel (retinol (RE) serum-free and feeder-free) system for the successful culture of adult germline stem cells from adult Kunming mice (8-24 weeks) testis. The isolated mGSCs cultured in RE serum-free and feeder-free medium maintained the typical morphology of undifferentiated embryonic stem cells (ESCs), and they proliferated well in RE medium analyzed by proliferation assay, RT-PCR, microarray, and Western blotting. These cells also showed typical properties of ESCs (alkaline phosphatase (AP) positive, expressions of Oct4, Sox2, Nanog, and SSEA1, with the capacity to form teratomas and differentiate into various types of cells within three germ layers). Taken together, we conclude that RE promotes the self-renewal of mGSCs and maintains the pluripotency of mGSCs, the RE serum-free and feeder-free system may be useful for the culture of pluripotent stem cell lines from adult testis tissues, which provides a new resource for tissue engineering and therapy for infertility.
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Derivation of male germ cell-like lineage from human fetal bone marrow stem cells.
Reprod. Biomed. Online
PUBLISHED: 07-04-2009
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Mesenchymal stem cells derived from bone marrow are a well characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of cell types. Reports have shown that murine and human embryonic stem cells can differentiate into primordial germ cells and then to early gametes. Evidence has indicated that some adult stem cells also have the potential to differentiate into germ cells. Currently, there are no reports on directed differentiation of human mesenchymal stem cells into germ cells. This study investigated the ability of retinoic acid and testicular extracts to induce human bone marrow stem cells (hBMSC) to differentiate into male germ cells. It was found that a small population of hBMSC seem to transdifferentiate into male germ cell-like cells. These cells expressed early germ cell markers OCT4, STELLA, NANOG and VASA, and male germ-ceil-specific markers such as DAZL, TH2, c-kit, beta(1)-integrin, ACR, PRMl, FSHR, STRA8 and SCP3, as analysed by reverse transcription-polymerase chain reaction and immunohistochemistry. These results demonstrated that hBMSC may differentiate into male germ cells and the same could be used as a potential source of cells for reproductive toxicological studies.
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Characterization of mesenchymal stem cells (MSCs) from human fetal lung: potential differentiation of germ cells.
Tissue Cell
PUBLISHED: 05-10-2009
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Pluripotent mesenchymal stem-like cell lines were established from lungs of 3-4 months old aborted fetus. The cells present the high ex vivo expansion potential of MSC, a typical fibroblast-like morphology and proliferate up to 15 passages without displaying clear changes in morphology. Immunological localization and flow cytometry analyses showed that these cells are positive for OCT4, c-Kit, CD11, CD29, CD44, telomerase, CD106, CD105, CD166, and SSEA1, weakly expression or negative for SSEA1, SSEA3, SSEA4, CD34, CD105 and CD106. These cells can give rise to the adipogenic as evidenced by accumulation of lipid-rich vacuoles within cells identified by Oil-red O when they were induced with 0.5 mM isobutylmethylxanthine, 200 microM indomethacin, 10(-6)M dexamethasone, and 10 microg/ml of insulin in high-glucose DMEM. Osteogenic lineage cells were generated in 0.1 microM dexamethasone, 50 microg/ml ascorbic acid, 10 mM beta-glycerophosphate, which are shaped as the osteoblastic morphology, expression of alkaline phosphatase (AP), and the formation of a mineralized extracellular matrix identified by Alizarin Red staining. Neural cells are observed when the cultures were induced with 2-mercapometal, which are positive for nestin, NF-100, MBP and GFAP. Additionally, embryoid bodies (EBs) and sperm like cells are obtained in vitro differentiation of these lung MSCs induced with 10(-5)M retinoic acid (RA). These results demonstrated that these MSCs are pluripotent and may provide an in vitro model to study germ-cell formation and also as a potential source of sperms for male infertility.
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Platelet-derived growth factor promotes the proliferation of human umbilical cord-derived mesenchymal stem cells.
Cell Biochem. Funct.
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This study was designed to investigate the effect of platelet-derived growth factor (PDGF) on the proliferation of human umbilical cord mesenchymal stem cells (UC-MSCs) and further explore the mechanism of PDGF in promoting the proliferation of UC-MSCs. The human UC-MSCs were treated with different concentrations of PDGF, and the effects were evaluated by counting the cell number, the cell viability, the expression of PDGF receptors analyzed by RT-PCR, and the detection of the gene expression of cell proliferation, cell cycle and pluripotency, and Brdu assay by immunofluorescent staining and Quantitative real-time (QRT-PCR). The results showed that PDGF could promote the proliferation of UC-MSCs in vitro in a dose-dependent way, and 10 to 50 ng/ml PDGF had a significant proliferation effect on UC-MSCs; the most obvious concentration was 50 ng/ml. Significant inhibition on the proliferation of UC-MSCs was observed when the concentration of PDGF was higher than 100 ng/ml, and all cells died when the concentration reached 200 ng/ml PDGF. The PDGF-treated cells had stronger proliferation and antiapoptotic capacity than the control group by Brdu staining. The expression of the proliferation-related genes C-MYC, PCNA and TERT and cell cycle-related genes cyclin A, cyclin 1 and CDK2 were up-regulated in PDGF medium compared with control. However, pluripotent gene OCT4 was not significantly different between cells cultured in PDGF and cells analyzed by immunofluorescence and QRT-PCR. The PDGF could promote the proliferation of human UC-MSCs in vitro.
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