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  • Kumar, S.  (5)
  • Biodiversity Research  (5)
  • 1
    Online Resource
    Online Resource
    120 IDENTIFICATION OF THE RELATIVELY PREDOMINANT BUFFALO INTERFERON tau ISOFORM AND ITS EXPRESSION IN ESCHERICHIA COLI
    CSIRO Publishing ; 2014
    In:  Reproduction, Fertility and Development Vol. 26, No. 1 ( 2014), p. 174-
    Details
    In: Reproduction, Fertility and Development, CSIRO Publishing, Vol. 26, No. 1 ( 2014), p. 174-
    Abstract: The maternal pregnancy recognition factor interferon tau (IFN-τ) is expressed in multiple isoforms in all pecoran ruminant species. Interferon-τ, as the first pregnancy signaling molecule, performs a significant role in implantation as well as establishment of pregnancy. Due to low reproductive efficiency of buffalo compared with bovine and IFN-τ being the key molecule of reproductive physiology in ruminants, the objective of our study was framed to identify the various IFN-τ transcripts in buffalo embryonic trophoblast cells, to know their relative abundance to identify the relatively predominant isoform, and lastly to clone and express it in a heterologous host. Following total cellular RNA extraction from primary trophectodermal cells, RT-PCR was performed using gene-specific primers designed against known bovine IFN-τ sequence. Cloning of the amplified product and screening of the recombinant colonies gave 13 distinct cDNA variants that encoded for 8 distinct buffalo IFN-τ isoforms. These buffalo IFN-τ isoforms have a greater nucleotide and amino acid homology with caprine IFN-τ (98–100% and 96–100%) than ovine (94–97% and 90–95%) and bovine (89.6–90.6% and 82–86%), respectively. The novel buffalo IFN-τ isoforms showed pronounced nucleotide and amino acid sequence identity with one another (99.1–99.8% and 98–99%) but only moderate identity with previously identified buffalo IFN-τ (90–92% and 82–86%). All the 13 transcript sequences were accepted in GenBank. Out of 8 isoforms, buffalo IFN-τ1 has been found to be the relatively predominant, which was subcloned into expression vector pET 22b without signal sequence from pJET cloning vector and expressed in competent BL21 (DE3) Escherichia coli strain. Expression of the recombinant protein in soluble form was induced by isopropyl β-D-1-thiogalactopyranoside (0.1 mM) at 30°C for 6 h. The recombinant BuIFN-τ obtained was confirmed by Western blot using anti-HIS antibody. A new 20-kDa protein was detected coinciding the molecular weight of IFN-τ reported earlier in literature. In conclusion, the current study revealed that there are 8 different isoforms of IFN-τ that are expressed in trophectodermal out-growths during early pregnancy of buffalo. Predominantly found isoform IFN-τ 1 was expressed in pET 22b vector, and recombinant soluble protein was confirmed by Western blot.
    Type of Medium: Online Resource
    ISSN: 1031-3613
    URL: Article
    DOI: 10.1071/RDv26n1Ab120
    Language: English
    Publisher: CSIRO Publishing
    Publication Date: 2014
    SSG: 12
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  • 2
    Online Resource
    Online Resource
    199 GERM-CELL-LIKE CELLS GENERATION FROM GOAT INDUCED PLURIPOTENT STEM CELLS
    CSIRO Publishing ; 2014
    In:  Reproduction, Fertility and Development Vol. 26, No. 1 ( 2014), p. 214-
    Details
    In: Reproduction, Fertility and Development, CSIRO Publishing, Vol. 26, No. 1 ( 2014), p. 214-
    Abstract: Primordial germ cells (PGCs) generated from embryonic stem (ES) cells in different species may be an alternative approach to dealing with the worldwide problem of increasing female infertility. Reprogramming of fibroblasts into induced pluripotent stem cells has been achieved by overexpression of different transcription factors. Here, we report the generation of female goat germ cells from goat induced pluripotent stems cells (giPSC). Goat induced pluripotent stem cells (giPSC) were produced by transduction of adult female goat fibroblast cells with Oct4, Sox2, and Nanog lentiviral particles and further sub-cultured on fibroblast feeder layers. GiPSC were characterised by different methods. These iPSC were found to express alkaline phosphatase, SSEA1, SSEA4, Tra-1–81, and Tra-1–60 surface markers. However, SSEA3 was not observed in giPSC. GiPSC also expressed Oct4, Nanog, and Sox2. Along with Oct4, Nanog, and Sox2, the expression of different transcription factors such as Cdx1, Dapp5, Dax1, Ecat, Eras, Fgf4, Gata6, Lin28, Rex1, and Utf1 was confirmed by RT-PCR. GiPSC were in vitro differentiated and three germ layers were characterised by immunostaining of Gata4 for endoderm, α-Actinin for mesoderm, and β-III tubulin for ectoderm and RT-PCR analysis of GATA4, α-Actinin and BMP4. IPSCs were directed differentiated into germ cells using retinoic acid and bone morphogenetic protein 4 without the inactivation of exogenous factors as these are also required for germ cells development. Differentiated germ cells were characterised by immunostaining against VASA and Dazl proteins. RT–PCR assay was performed for Dazl, Nanog, Nanos1, PUM8, SCP3, Stella, and VASA genes expression. Quantitative PCR was also performed for detection of VASA and Dazl expression during the course of germ cell differentiation. Flow-cytometric analysis of differentiated germ cells was confirmed the presence of germ cells in population of differentiated giPSC. Oocytes/ova-like structures, which were comparable to natural goat oocytes, were observed under scanning electron microscope (SEM). Cumulus–oocyte complex like structure was observed, which was further used for SEM. The study concluded that adult female goat fibroblast cells can be reprogrammed into induced pluripotent stem cells using ectopic expression of Oct4, Nanog, and Sox2 genes and the germ-cells-like cells generated from reprogrammed giPSC could be differentiated into goat oocytes/ova-like structure which have immense applications in human and animal reproduction.
    Type of Medium: Online Resource
    ISSN: 1031-3613
    URL: Article
    DOI: 10.1071/RDv26n1Ab199
    Language: English
    Publisher: CSIRO Publishing
    Publication Date: 2014
    SSG: 12
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  • 3
    Online Resource
    Online Resource
    198 ISOLATION, CHARACTERIZATION, AND IN VITRO DIFFERENTIATION OF GOAT ADIPOSE-TISSUE-DERIVED MESENCHYMAL STEM CELLS INTO PANCREATIC ISLETS-LIKE CELLS
    CSIRO Publishing ; 2014
    In:  Reproduction, Fertility and Development Vol. 26, No. 1 ( 2014), p. 213-
    Details
    In: Reproduction, Fertility and Development, CSIRO Publishing, Vol. 26, No. 1 ( 2014), p. 213-
    Abstract: The present study was carried out for isolation of goat (Capra hircus) adipose-tissue-derived stem cells (gADSCs) from adipose tissue, their characterization, and in vitro differentiation of gADSCs into pancreatic islets-like cells by giving conditioned medium. Goat ADSCs were isolated from goat adipose tissue by the enzymatic digestion method and were enriched by filtering through a 41-μm filter. Thus, filtered cells resuspended in a cell culture flask containing growth enriching medium and cultured in 5% CO2 in air at 38.5°C. Goat ADSCs were characterised by amplification of mesenchymal stem cell specific markers i.e. CD29, CD34, CD44, CD90, and CD166 as positive markers and CD41 and CD71 as negative markers. Immunocytochemistry of mesenchymal stem cell was also carried out with specific markers CD44 and CD90. Goat ADSCs were further characterised by in vitro differentiating them into osteocytes, chondrocytes, and adipocytes. For in vitro differentiation of gADSCs into osteocytes gADSCs were supplemented with conditioned medium i.e. DMEM containing fetal bovine serum (FBS), dexamethazone, B-glycerol phosphate and L-ascorbic acid. Osteogenic differentiation was confirmed by positive Alizarin red S staining and amplification of Osteopontin and Collagen I genes. For differentiation into chondrocytes cells, gADSCs were incubated in DMEM/F12 containing dexamethazone, ITX, BMP-4, and FBS for 21 days. Differentiated cells were confirmed by positive Safranin O staining and expression of chondrocytes specific Collagen III and Aggrecan genes. For adipogenesis, gADSCs were incubated with DMEM/F12 containing FBS, dexamethasone, and ITX and differentiated cells were confirmed by positive Oil Red O staining and amplification of adipocytes specific genes i.e. LPL, PPRγ and PPRα. For in-vitro differentiation gADSCs into pancreatic islets-like cells on the third or fourth passage gADSCs were incubated in conditioned medium containing serum-free DMEM/F12 medium with glucose (17.5 mM) in the presence of nicotinamide (10 mM), activin-A (2 nM), exendin-4 (10 nM), pentagastrin (10 nM), retinoic acid (10 μM) and mercaptoethanol (20 μM). The in vitro differentiation gADSCs into pancreatic islets-like cells was confirmed by amplification of pancreatic endoderm specific genes i.e. igf-1, sst, ngn3, pdx-1, isl-1, c-kit, thy-1, and Glut-2, and no expression was detected for above endoderm specific genes in undifferentiated gADSCs. Pancreatic islets-like cells were further characterised by immunostaining and Western blotting of Pdx-1, insulin, and Islets-1 specific protein. It could be concluded that gADSCs was differentiated into different lineages and secretory insulin was produced from pancreatic islets-like cells.
    Type of Medium: Online Resource
    ISSN: 1031-3613
    URL: Article
    DOI: 10.1071/RDv26n1Ab198
    Language: English
    Publisher: CSIRO Publishing
    Publication Date: 2014
    SSG: 12
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  • 4
    Online Resource
    Online Resource
    204 ISOLATION, CHARACTERIZATION, AND DIFFERENTIATION OF ADIPOSE TISSUE DERIVED MESENCHYMAL STEM CELLS: AN AUTOLOGOUS TRANSPLANTATION TO PATIENTS
    CSIRO Publishing ; 2014
    In:  Reproduction, Fertility and Development Vol. 26, No. 1 ( 2014), p. 216-
    Details
    In: Reproduction, Fertility and Development, CSIRO Publishing, Vol. 26, No. 1 ( 2014), p. 216-
    Abstract: Adult stem cells derived from all possible sources of livestock serve as the best possible alternative to embryonic stem cells. The discovery of mesenchymal stem cells has provided the new horizon to stem cell therapy. Adipose tissue derived mesenchymal stem cell (ADSCs), an easy source of adult stem cell has created a lot of interest among researchers as patient specific treatment and autologous transplantation in animals is becoming a viable option. The proposed study was carried out for 1) isolation of ADSCs from dogs, suffering from hip dysplasia or from paraplegia, 2) ADSC characterisation and in vitro differentiation ability into osteocytes, chondrocytes, adipocytes and neurocytes specific cells. Adipose tissues were collected from belly/umbilical cord region. ADSCs were isolated by enzymatic digestion method followed by enriching through a 41 μm filter. Filtered cells were then resuspended in cell culture flasks containing growth enriching medium and cultured in 5% CO2 in air at 37°C for 5 days. ADSCs were characterised by amplification of mesenchymal stem cell specific markers i.e. CD29, CD44, CD90, and CD166 and by immunocytochemistry of mesenchymal stem cell specific protein i.e. CD44 and CD90. ADSCs were further in vitro differentiated. ADSCs derived osteocytes, chondrocytes, and adipocytes were validated through the amplification of specific markers of osteocytes (Osteopontin, Collagen I); chondrocytes (Aggrecan and Collagen II) and adipocytes (LPL, PPARα, PPARγ). Dog ADSCs were further autogenic transplanted into hip dysplasia and paraplegic patients. These patients recovered well one month from transplantation and were able to move freely. It may be concluded that these findings may have implications for defining the physiological roles of ADSCs in arthritis; orthopaedic ailments, joint regeneration, neuronal disorders, and several other applications leading to novel therapeutic opportunities.
    Type of Medium: Online Resource
    ISSN: 1031-3613
    URL: Article
    DOI: 10.1071/RDv26n1Ab204
    Language: English
    Publisher: CSIRO Publishing
    Publication Date: 2014
    SSG: 12
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  • 5
    Online Resource
    Online Resource
    A gravitational-wave standard siren measurement of the Hubble constant
    Springer Science and Business Media LLC ; 2017
    In:  Nature Vol. 551, No. 7678 ( 2017-11-02), p. 85-88
    Details
    In: Nature, Springer Science and Business Media LLC, Vol. 551, No. 7678 ( 2017-11-02), p. 85-88
    Type of Medium: Online Resource
    ISSN: 0028-0836 , 1476-4687
    URL: Article
    DOI: 10.1038/nature24471
    RVK:
    TA 1000
    RVK:
    UA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2017
    detail.hit.zdb_id: 120714-3
    detail.hit.zdb_id: 1413423-8
    SSG: 11
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