scholarly journals Creation of cloned pig embryos using contact-inhibited or serum-starved fibroblast cells analysed intravitam for apoptosis occurrence / Uzyskiwanie klonalnych zarodków świni z wykorzystaniem komórek fibroblastycznych poddanych inhibicji kontaktowej lub deprywacji troficznej oraz analizowanych przyżyciowo w kierunku apoptozy

2013 ◽  
Vol 13 (2) ◽  
pp. 275-293 ◽  
Author(s):  
Marcin Samiec ◽  
Maria Skrzyszowska ◽  
Jolanta Opiela
Keyword(s):  
Dermal Fibroblast ◽  
Somatic Cells ◽  
Fibroblast Cell ◽  
Serum Starvation ◽  
Fibroblast Cells ◽  
Blastocyst Formation ◽  
Cell Nuclei ◽  
Donor Cells ◽  
Cloned Pig

Abstract Somatic cell cloning efficiency is determined by many factors. One of the most important factors is the structure-functional quality of nuclear donor cells. Morphologic criteria that have been used to date for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Biochemical and biophysical changes that are one of the earliest symptoms in the transduction of apoptotic signal may be not reflected in the morphologic changes of somatic cells. For this reason, adult cutaneous or foetal fibroblast cells that, in our experiments, provided the source of genomic DNA for the cloning procedure had been previously analysed for biochemical and biophysical proapoptotic alterations with the use of live-DNA (YO-PRO-1) and plasma membrane (Annexin V-eGFP) fluorescent markers. In Groups IA and IB, the generation of nucleartransferred (NT) embryos using non-apoptotic/non-necrotic contact-inhibited or serum-starved adult cutaneous fibroblast cells yielded the morula and blastocyst formation rates of 125/231 (54.1%) and 68/231 (29.4%) or 99/237 (41.8%) and 43/237 (18.1%), respectively. In Groups IIA and IIB, the frequencies of embryos reconstituted with non-apoptotic/non-necrotic contact-inhibited or serum-starved foetal fibroblast cell nuclei that reached the morula and blastocyst stages were 171/245 (69.8%) and 97/245 (39.6%) or 132/227 (58.1%) and 63/227 (27.8%), respectively. In conclusion, contact inhibition of migration and proliferative activity among the subpopulations of adult dermal fibroblast cells and foetal fibroblast cells resulted in considerably higher morula and blastocyst formation rates of in vitro cultured cloned pig embryos compared to serum starvation of either type of fibroblast cell line. Moreover, irrespective of the methods applied to artificially synchronize the mitotic cycle of nuclear donor cells at the G0/G1 phases, developmental abilities to reach the morula/blastocyst stages were significantly higher for porcine NT embryos that had been reconstructed with non-apoptotic/non-necrotic foetal fibroblast cells than those for NT embryos that had been reconstructed with non-apoptotic/non-necrotic adult dermal fibroblast cells. To our knowledge, the generation of cloned pig embryos using abattoir-derived oocytes receiving cell nuclei descended from contact-inhibited or serum-deprived somatic cells undergoing comprehensive vital diagnostics for the absence of biochemical and biophysical proapoptotic alterations within their plasmalemmas has not been reported so far.

51 DEVELOPMENT OF EQUINE CLONED EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES RECEIVING ADULT DERMAL FIBROBLAST CELL NUCLEI

2009 ◽  
Vol 21 (1) ◽  
pp. 125
Author(s):  
M. Skrzyszowska ◽  
M. Samiec ◽  
W. Mlodawska ◽  
J. Kochan ◽  
A. Okolski ◽  
...  
Keyword(s):  
Dermal Fibroblast ◽  
Fibroblast Cell ◽  
Vero Cells ◽  
Contact Inhibition ◽  
Fibroblast Cells ◽  
Cell Nuclei ◽  
Metaphase Chromosomes ◽  
Condensed Chromosome ◽  
Donor Cells

The purpose of our study was to determine the in vitro developmental competences of equine NT embryos reconstructed with adult dermal fibroblast cells. Frozen/thawed fibroblast cells, whose mitotic cycle had been synchronized at G1/G0 stages through a contact inhibition of their migration and proliferative activity under total confluency, were used as a source of nuclear donor cells in the somatic cell cloning procedure. In vitro-matured oocytes were used as recipient cells for fibroblast cell nuclei. The compact cumulus–oocyte complexes (cpCOCs) were collected from abattoir-derived mare ovaries and selected for in vitro maturation. The cpCOCs were cultured in TC-199 medium supplemented with 5 mU mL–1 follicle-stimulating hormone (FSH), 10% fetal bovine serum (FBS) and 75 μg mL–1 kanamycin monosulfate (kanamycin A) for 30 h at 38.2°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Cumulus-denuded in vitro-matured oocytes were incubated in the maturation medium supplemented with 0.4 μg mL–1 demecolcine for 40 min. The treated oocytes were subsequently transferred into TC-199 medium containing 4 mg mL–1 BSA-V and 5 μg mL–1 cytochalasin B. Metaphase chromosomes, which had been allocated into the chemically-induced protrusion of the plasma membrane, were removed microsurgically. The chemically-assisted enucleation was accomplished by gently aspirating the ooplasmic cone, which contained the condensed chromosome mass, with the aid of a beveled micropipette. The single nuclear donor cells were inserted into perivitelline space of previously enucleated oocytes. Fibroblast cell-ooplast couplets were fused with two consecutive DC pulses of 2.4 kV cm–1 for 30 μs. After a 1.5-h delay, nuclear transfer-derived oocytes were chemically activated by exposure to 5 μm L–1 calcium ionomycin for 5 to 7 min, followed by their incubation in B2 medium with addition of 2 mm L–1 6-dimethylaminopurine (6-DMAP) for 4 h. Reconstructed embryos were in vitro cultured in B2 medium for 2 days. Afterwards, cleaved embryos were co-cultured with Vero cells in B2 medium supplemented with 10% FBS for 5 to 6 days up to morula/blastocyst stages. From among 88 in vitro cultured cpCOCs, 55 (62.5%) acquired meiotic nuclear and cytoplasmic maturity state after reaching the Metaphase II stage. A total of 55 enucleated oocytes underwent reconstruction and 44/55 (80.0%) were successfully fused with nuclear donor cells. Out of 44 cultured NT embryos, 21 (47.7%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 6/44 (13.6%) and 3/44 (6.8%), respectively. In conclusion, the cell nuclei of in vitro cultured adult dermal fibroblast cells, which had undergone the contact inhibition, were able to direct the preimplantation development of equine cloned embryos to morula and blastocyst stages. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

79 PSEUDOPHYSIOLOGICAL TRANSCOMPLEMENTARY ACTIVATION OF GOAT OOCYTES RECEIVING ADULT EAR CUTANEOUS FIBROBLAST CELL NUCLEI

2010 ◽  
Vol 22 (1) ◽  
pp. 198
Author(s):  
M. Skrzyszowska ◽  
M. Samiec
Keyword(s):  
Somatic Cell ◽  
Plasma Membranes ◽  
Dermal Fibroblast ◽  
Fibroblast Cell ◽  
Vero Cells ◽  
Atmospheric Conditions ◽  
Cell Nuclei ◽  
Developmental Potential ◽  
Water Saturated

The aim of the study was to determine the in vitro developmental potential of caprine cloned embryos following pseudophysiological (transcytoplasmic) transcomplementary activation of oocytes reconstructed with ear skin-derived fibroblast cell nuclei. The source of nuclear recipient cells were IVM doe oocytes. The reconstruction of the previously enucleated oocytes (i.e. ooplasts) was performed by microinjection of either the somatic cell-derived karyoplasts or intact whole tiny nuclear donor cells directly into the cytoplasm. The reconstructed oocytes were incubated in Upgraded B2 INRA medium for 30 min to 1 h before their pseudophysiological activation. The activation was achieved by electrofusion of clonal cybrids with the allogeneic cytoplasts isolated from caprine IVF-created zygotes, which led to the formation of triple allocytoplasmic hybrids (allocybrids). These originate from 3 sources: (1) homologous whole nuclear donor fibroblast cells or their karyoplasts; (2) enucleated oocytes (ooplasts), and (3) zygote-derived cytoplasts. Single zygote-descended cytoplasts (the so-called zygoplasts) were inserted into the perivitelline space of previously reconstituted oocytes. The resulting zygoplast-clonal cybrid couplets were subsequently subjected to electrofusion, which was induced by application of a single DC pulse of 2.4 kV cm-1 for 15 μs. The electrofusion of zygoplast and reconstructed oocyte plasma membranes occurred in an isotonic dielectric solution deprived of Ca2+ ions. The transcytoplasmically activated clonal cybrids were cultured in vitro in Upgraded B2 INRA medium for 48 h at 38.5°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Afterward, cleaved embryos were co-cultured with Vero cells in medium supplemented with 10% fetal bovine serum for an additional 96 to 144 h up to morula and blastocyst stages under the same thermal and atmospheric conditions. A total of 53/78 (67.9%) oocytes reconstructed with fibroblast cell nuclei were successfully fused with zygoplasts. From among 53 cultured cloned embryos, 34 (64.2%) cleaved. The rates of embryos that reached the morula and blastocyst stages were 21/53 (39.6%) and 11/53 (20.8%), respectively. In conclusion, the relatively high percentages of morulae and blastocysts were noticed among in vitro-cultured caprine cloned embryos produced by the strategy of pseudophysiological transcytoplasmic activation of oocytes reconstructed with adult dermal fibroblast cell nuclei. Therefore, the use of cytoplasmic components originating from zygotes as the stimuli for activation of nuclear-transferred oocytes appeared to be an effective procedure in the generation of goat blastocysts by somatic cell cloning.

Cellular reprogramming of adult goat fibroblast: Toward pluripotency

2016 ◽  
Author(s):  
Dharmendra Kumar ◽  
Rakesh Ranjan ◽  
Ajit P. Singh ◽  
Bikash C Sarkhel
Keyword(s):  
Cell Lines ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Fibroblast Cell ◽  
Cellular Reprogramming ◽  
Serum Starvation ◽  
Methyl Transferase ◽  
Donor Cells ◽  
Key Factor ◽  
Gene Transcripts

Cellular reprogramming erases the epigenetic constraints of somatic cells genome and thus considered as key factor for success of somatic cell nuclear transfer technology. To achieve the reprogramming, different strategies are used which are mostly based on arresting the cell cycle at G0 or G1 stage. The present study was based on molecular investigation of reprogrammed cells for expression of pluripotent genes that are crucial for development of cloned embryos. The fibroblast cell lines were treated by four methods to induce cellular reprogramming viz., serum starvation, Roscovitin, aphidicolin and overconfluent. These treated cell lines were used for quantification of pluripotent gene transcripts by using real time PCR machine. The results showed that the relative expression of different pluripotent genes as Oct-4 and Nanog along with DNA methyl transferase gene (Dnmt-1) was observed in four treated cells. In case of normal cells, only Dnmt-1 gene was expressed, but pluripotent genes were not expressed at detection level. The expression of pluripotent genes in the donor cells prior to nuclear transfer have significant impact on cloning as because it facilitates the expression of that gene in the resulting embryo after nuclear transfer. The finding of this study may be extended for stem cell generation as it showed that pluripotent genes could be induced in the somatic cells without any transgenic incorporation.

scholarly journals Assessment of in Vitro Developmental Capacity of Porcine Nuclear-Transferred Embryos Reconstituted with Cumulus Oophorus Cells Undergoing Vital Diagnostics for Apoptosis Detection / Ocena zdolności rozwojowych in vitro klonalnych zarodków świni rekonstytuowanych z jąder komórek wzgórka jajonośnego poddawanych przyżyciowej diagnostyce w kierunku wykrywania apoptozy

2013 ◽  
Vol 13 (3) ◽  
pp. 513-529 ◽  
Author(s):  
Marcin Samiec ◽  
Maria Skrzyszowska
Keyword(s):  
Plasma Membranes ◽  
Dermal Fibroblast ◽  
Annexin V ◽  
Cumulus Cells ◽  
Fibroblast Cells ◽  
Developmental Potential ◽  
Cumulus Oophorus ◽  
Group I ◽  
Cloned Pig

Abstract The objective of the current investigation was to extensively compare the in vitro developmental capabilities between cloned pig embryos reconstructed with the cell nuclei of either cumulus oophorus cells or adult dermal fibroblast cells that were both evaluated as non-apoptotic on the basis of YO-PRO-1- and Annexin V-eGFP-mediated vital analysis for programmed cell suicide. In Group I, the competences of nuclear-transferred (NT) embryos that were derived from non-apoptotic/ non-necrotic (i.e., YO-PRO-1- and Annexin V-eGFP-negative) cumulus cells to complete their development to the morula and blastocyst stages were maintained at the proportions of 155/364 (42.6%) and 54/364 (14.8%), respectively. In Group II, NT embryos that were reconstituted with non-apoptotic and/or non-necrotic adult cutaneous fibroblast cells developed to the morula and blastocyst stages at the rates of 207/358 (57.8%) and 110/358 (30.7%), respectively. Although the in vitro developmental potential of porcine NT embryos derived from non-apoptotic/non-necrotic cumulus cells was significantly lower (P<0.001) than that of NT embryos reconstructed with adult dermal fibroblast cells, the obtained morula/blastocyst formation rates turned out to be considerably higher as compared to the rates reported by other investigators. Altogether, to our knowledge, the comprehensive research aimed at the determination of preimplantation developmental outcomes of cloned pig embryos produced using nuclear donor somatic cells of different provenance (cumulus oophorus cells or adult cutaneous fibroblast cells) that were vitally diagnosed for the lack of proapoptotic transformations in their plasma membranes has not yet been accomplished.

55 LIVE PLASMA MEMBRANE ANALYSIS OF EARLY APOPTOTIC CELL DEATH IN PORCINE ADULT DERMAL FIBROBLASTS PRIOR TO SOMATIC CELL CLONING

2008 ◽  
Vol 20 (1) ◽  
pp. 108
Author(s):  
M. Skrzyszowska ◽  
M. Samiec
Keyword(s):  
Plasma Membrane ◽  
Somatic Cell ◽  
Dermal Fibroblast ◽  
Chemical Activation ◽  
Annexin V ◽  
Fibroblast Cells ◽  
Apoptotic Cells ◽  
Cell Cloning ◽  
Donor Cells

The aim of our study was to determine the in vitro developmental capability of porcine nuclear-transferred (NT) embryos reconstructed with adult dermal fibroblast cells, which had been analyzed for apoptosis by live plasma membrane fluorescent labelling. Frozen/thawed fibroblasts, which had been in vitro cultured to confluency, were used for analysis. To detect the early apoptotic changes in the plasma membrane involving the externalization of phosphatidylserine molecules and the subsequent loss of lipid composition asymmetry, the fibroblasts were tagged using a conjugate of annexinV with enhanced green fluorescent protein (eGFP). In the somatic cell cloning procedure, enucleated in vitro-matured oocytes were reconstituted with non-apoptotic dermal fibroblast cell nuclei. Afterwards, NT-derived oocytes were stimulated with a combination of electrical and chemical activation. Simultaneous fusion and electrical activation were induced by application of two successive DC pulses of 1.2 kV cm–1 for 60 �s. A two-step chemical activation procedure was initiated after a 1.5–2 h delay. The cybrids were exposed to 15 µm calcium ionomycin for 5 to 7 min and then incubated in the culture medium supplemented with 10 µg mL–1 cycloheximide for 3 h. Reconstructed embryos were in vitro cultured in NCSU-23 medium for 6–7 days. Fluorescence analysis of the adult dermal fibroblast cells revealed that a relatively high proportion of donor cells exhibited proapoptotic changes in the plasma membrane. The percentage of late apoptotic cells with advanced morphological changes did not exceed 30%. Moreover, an extremely low rate (ranging from 0 to 2%) of early apoptotic cells, with a morphologically normal, i.e., smooth (non-corrugated) and intact (non-blebbing), plasmolemma but which emitted the green eGFP-derived chemiluminescence, was detected. A total of 219 enucleated oocytes were subjected to reconstruction and 185/219 (84.5%) were successfully fused with non-apoptotic nuclear donor cells. Out of 185 cultured NT embryos, 108 (58.4%) cleaved. The frequencies of cloned embryos, that reached the morula and blastocyst stages, were 84/185 (45.4%) and 26/185 (14.0%), respectively. In conclusion, annexin V-eGFP is a sensitive method able to detect the early phases of apoptosis in cultured adult dermal fibroblast cells, because it identified that very small proportion of morphologically normal cells (without shrinkage of the plasmolemma) that also emitted the annexin V-eGFP-derived biochemiluminescence. Nonetheless, the probability of their random erroneous selection for somatic cell cloning appears to be extremely low. It was also found that the preimplantation developmental potential of NT embryos originating from non-apoptotic adult dermal fibroblast cells is relatively high. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

scholarly journals 65 PRODUCTION OF PORCINE NUCLEAR TRANSFER EMBRYOS USING FETAL FIBROBLAST CELLS ANALYZED ON APOPTOSIS

2005 ◽  
Vol 17 (2) ◽  
pp. 182 ◽  
Author(s):  
M. Skrzyszowska ◽  
M. Samiec
Keyword(s):  
Nuclear Transfer ◽  
Fibroblast Cell ◽  
State Committee ◽  
Fibroblast Cells ◽  
Developmental Potential ◽  
Fetal Fibroblast ◽  
Functional Quality ◽  
Donor Cells ◽  
Morphological Transformations

One of the most important factors that determine the developmental potential of mammalian cloned embryos is the structuro-functional quality of nuclear donor cells. Biochemical changes that are some of the earliest symptoms of apoptosis signal transduction are not reflected in the morphological features of somatic cells. Therefore, an appropriate system of cell selection would enable the sorting of donor nuclei with high morphological and biochemical susceptibility to somatic cloning. The aim of our study was to examine the in vitro developmental competencies of porcine nuclear transfer (NT) embryos reconstructed with fetal fibroblast cells that had been analyzed for apoptosis by live-fluorescent labelling. Frozen/thawed fetal fibroblast cells, which had been in vitro-cultured to a confluent state, were used for analysis. To detect the early apoptotic changes in the fibroblast cells, a single cell suspension of nuclear donor cells was subjected to dyeing with live-DNA green fluorochrome YO-PRO-1. The recipient cells were in vitro-matured oocytes. Maternal chromosomes were removed by a chemically assisted microsurgical technique. Then, single nuclear donor cells were inserted into the perivitelline space of enucleated oocytes. Fibroblast cell-ooplast couplets were simultaneously fused and activated with two consecutive DC pulses of 1.2 kV/cm for 60 μs. Reconstructed embryos were in vitro cultured in 50-μL drops of NCSU-23 medium supplemented with 0.4% BSA-V for 6 to 7 days at 38.5°C in a humidified atmosphere of 5% CO2 and 95% air. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. After fluorescent analysis of approximately 50 different random samples collected from the population of fetal fibroblast cells, that had been labelled with YO-PRO-1 dye, it was found that a relatively high proportion of donor cells revealed ultrastructural apoptotic changes. The percentage of late apoptotic cells with advanced morphological transformations was about 40% of the total pool of the fibroblast cells. A total of 262/270 (97.0%) enucleated oocytes were subjected to reconstruction and 141/262 (53.8%) were successfully fused with non-apoptotic nuclear donor cells. Following the simultaneous fusion/activation protocol, reconstituted oocytes were selected for in vitro culture. Out of 262, 133 (50.8%) cultured NT embryos cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 48/133 (36.0%) and 10/133 (7.5%), respectively. In conclusion, morphology is a sufficient selection factor for detection of apoptosis in the cultured (confluent) fetal fibroblast cells to be used for cloning. Moreover, it was found that YO-PRO-1 fluorochrome may be not able to detect the early phases of apoptosis, because only the morphologically abnormal cells emitted the YO-PRO-1-derived fluorescence. This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-KBN-084/P06/2002/4.2 from years 2003 to 2005.

scholarly journals In Vitro Development of Porcine Nuclear-Transferred Embryos Derived from Fibroblast Cells Analysed Cytometrically for Apoptosis Incidence and Accuracy of Cell Cycle Synchronization at the G0/G1 Stages / Rozwój In Vitro Klonalnych Zarodków Świni Zrekonstruowanych Z Jąder Komórkowych Fibroblastów Cytometrycznie Analizowanych Pod Kątem Częstotliwości Występowania Śmierci Apoptotycznej I Efektywności Synchronizacji Cyklu Mitotycznego W Fazach G0/G1

2013 ◽  
Vol 13 (4) ◽  
pp. 735-752 ◽  
Author(s):  
Marcin Samiec ◽  
Maria Skrzyszowska ◽  
Michał Bochenek
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Mitotic Cycle ◽  
Apoptotic Cell ◽  
Fibroblast Cell ◽  
Contact Inhibition ◽  
Fibroblast Cells ◽  
Cell Nuclei ◽  
Flow Cytometric

Abstract The study was undertaken to examine whether various strategies, including contact inhibition and serum starvation, that were used for artificial synchronization of mitotic cycle of porcine fibroblast cell lines affect differently the distribution of cell cycle stage frequencies and the occurrence of apoptotic cell death in the analysed cell samples. In vitro cultured (contact-inhibited or serumstarved) somatic cells were subjected to flow cytometric diagnostics of mitotic cycle together with the detection of late-apoptotic cell fractions with hypodiploid number of nuclear DNA molecules. Moreover, impact of the methods applied to synchronize the cell division cycle of different types of nuclear donor fibroblast cells (adult cutaneous and foetal fibroblasts) on the preimplantation developmental outcomes of cloned pig embryos was investigated. The developmental capabilities of nuclear-transferred (NT) embryos that were reconstituted with contact-inhibited or serum-depleted adult cutaneous fibroblast cells to reach the morula and blastocyst stages remained at the levels of 169/278 (60.8%) and 76/278 (27.3%) or 121/265 (45.7%) and 46/265 (17.4%), respectively. The proportions of NT embryos originating from contact-inhibited or serum-deprived foetal fibroblast cells that completed their development to the morula and blastocyst stages were 223/296 (75.3%) and 108/296 (36.5%) or 165/261 (63.2%) and 67/261 (25.7%), respectively. In conclusion, the flow cytometric analysis of cultured porcine adult cutaneous and foetal fibroblast cells revealed the high efficiency of the artificial synchronization of mitotic cycle at the G0/G1 stages as a consequence of applying the methods of either contact inhibition or serum deprivation. For both types of fibroblast cells used to reconstruct the enucleated oocytes, the strategies that were utilized to synchronize the cell division cycle of nuclear donor cells considerably influenced the in vitro developmental abilities of NT pig embryos. Developmental competencies to reach the morula/blastocyst stages for cloned embryos that had been reconstructed with contact-inhibited or serum-starved foetal fibroblast cell nuclei were significantly higher than those for embryos that had been reconstructed with contact-inhibited or serum-starved adult cutaneous fibroblast cell nuclei.

86 CREATION OF PORCINE TRANSGENIC NUCLEAR-TRANSFERRED EMBRYOS RECONSTRUCTED WITH EGFP-EXPRESSING ADULT DERMAL FIBROBLAST CELLS ANALYZED ON APOPTOSIS

2007 ◽  
Vol 19 (1) ◽  
pp. 160 ◽  
Author(s):  
M. Skrzyszowska ◽  
M. Samiec ◽  
Z. Smorag ◽  
D. Lipinski ◽  
R. Slomski
Keyword(s):  
Fluorescent Protein ◽  
Dermal Fibroblast ◽  
Annexin V ◽  
Donor Cell ◽  
Fibroblast Cells ◽  
Egfp Gene ◽  
Morphological Criteria ◽  
Donor Cells ◽  
Fluorescent Tagging

The purpose of our study was to determine the in vitro developmental competences of porcine nuclear transfer (NT) embryos reconstructed with pWAPhGH-GFPBsd transgene-nucleofected gilt ear skin-descended fibroblast cells, which had been diagnosed on apoptosis through the live-plasma membrane fluorescent tagging. Frozen–thawed fibroblast cells, which had been in vitro-cultured up to a total confluency after 2–8 passages, were used for analysis. To detect the early apoptotic changes in the fibroblast cells, single nuclear donor cell suspension was labeled with the conjugate of Annexin V and eGFP protein. The source of recipient cells were in vitro-matured oocytes. Maternal chromosomes were eliminated by a chemically assisted microsurgical technique. Fibroblast cell–ooplast couplets were simultaneously fused and activated. Reconstructed embryos were cultured in NCSU-23/BSA/FBS medium for 6–7 days. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. After fluorescent analysis of adult dermal fibroblast cells, it was shown that a relatively high proportion (ranging from 20 to 30%) of donor cells exhibited ultrastructural late-apoptotic or necrotic changes. In contrast, from among the morphologically normal cells, an extremely low rate (ranging from 0 to 2%) of the cells emitted the Annexin V-eGFP-derived green fluorescence, but the other ones did not emit this biochemiluminescence. This suggests that the former subpopulation of the cells was early-apoptotic, and the latter was non-apoptotic. A total of 158 enucleated oocytes were successfully fused with non-apoptotic transgenic nuclear donor cells and intended to be in vitro-cultured. Out of 158 reconstructed oocytes, 106 (67.1%) NT embryos were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 48/158 (30.4%) and 21/158 (13.3%), respectively. In conclusion, the nucleofection efficiency of in vitro-cultured porcine dermal fibroblast cells as estimated by nuclear donor live-fluorescent evaluation based on the expression index of the eGFP reporter transgene was nearly 100%. Moreover, our results demonstrate that the morphological criteria commonly used for cell viability classification are a sufficient selection factor for qualitative evaluation of nuclear donor cells to somatic cell cloning. It was also found that porcine nuclear-transferred morulae and blastocysts exhibited an approximately 100% index of xenogeneic eGFP gene transcriptional activity, which revealed the live diagnostics of emission intensity for green fluorescent protein-derived biochemiluminescence. This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-MIN-005/P04/2002/6 from year 2003 to year 2006.

scholarly journals Tribological, biocompatibility, and antibiofilm properties of tungsten–germanium coating using magnetron sputtering

2021 ◽  
Vol 32 (1) ◽  
Author(s):  
Mustafa Şükrü Kurt ◽  
Mehmet Enes Arslan ◽  
Ayşenur Yazici ◽  
İlkan Mudu ◽  
Elif Arslan
Keyword(s):  
Cell Culture ◽  
Magnetron Sputtering ◽  
Cell Line ◽  
Borosilicate Glass ◽  
Chromosomal Abnormalities ◽  
Dermal Fibroblast ◽  
Fibroblast Cell ◽  
Antibiofilm Activity ◽  
Borosilicate Glasses

AbstractIn this study, borosilicate glass and 316 L stainless steel were coated with germanium (Ge) and tungsten (W) metals using the Magnetron Sputtering System. Surface structural, mechanical, and tribological properties of uncoated and coated samples were examined using SEM, X-ray diffraction (XRD), energy-dispersive spectroscopy, and tribometer. The XRD results showed that WGe2 chemical compound observed in (110) crystalline phase and exhibited a dense structure. According to the tribological analyses, the adhesion strength of the coated deposition on 316 L was obtained 32.8 N, and the mean coefficient of friction was around 0.3. Biocompatibility studies of coated metallic biomaterials were analyzed on fibroblast cell culture (Primary Dermal Fibroblast; Normal, Human, Adult (HDFa)) in vitro. Hoescht 33258 fluorescent staining was performed to investigate the cellular density and chromosomal abnormalities of the HDFa cell line on the borosilicate glasses coated with germanium–tungsten (W–Ge). Cell viabilities of HDFa cell line on each surface (W–Ge coated borosilicate glass, uncoated borosilicate glass, and cell culture plate surface) were analyzed by using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay. The antibiofilm activity of W–Ge coated borosilicate glass showed a significant reduction effect on Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATCC 27853) adherence compared to control groups. In the light of findings, tungsten and germanium, which are some of the most common industrial materials, were investigated as biocompatible and antimicrobial surface coatings and recommended as bio-implant materials for the first time.

42 DEVELOPMENT OF PIG EMBRYOS CLONED FROM DONOR CELLS TREATED WITH TRICHOSTATIN A

2008 ◽  
Vol 20 (1) ◽  
pp. 101 ◽  
Author(s):  
J. Li ◽  
Y. Du ◽  
P. M. Kragh ◽  
S. Purup ◽  
K. Villemoes ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Fibroblast Cells ◽  
Embryonic Genome ◽  
Donor Cells ◽  
Deacetylase Inhibitor ◽  
Vitro Development

Development to the blastocyst stage following nuclear transfer is dependent on the donor cell's ability to reprogram its genome to a totipotent state. Reprogramming of the transferred somatic nuclei must be completed by the time normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, Enright et al. (2003 Biol. Reprod. 69, 896–901) reported that in vitro development of cloned cow embryos was improved by treatment of donor cells with a histone deacetylase inhibitor, TrichostatinA (TSA). So far, there are no reports available for adult pig fibroblast cells treated with TSA. The objective of this study was to investigate whether the development of handmade cloned embryos in pig could be improved by using TSA-treated donor cells. Adult pig fibroblast cells were treated with 100, 150, or 200 nm TSA for 24 h, compared to untreated controls, and were then used as donor cells. The cells were electrofused with handmade enucleated pig oocytes separately and were activated with calcium ionophore and cycloheximide. They were subsequently cultured in porcine zygote medium 3 (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) using the well of the well system (WOW; Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Experiments were repeated 4 times and the data were analyzed with AVEDEV and t-test in Excel (Microsoft Excel 2007). The cleavage rates and the total cell numbers per blastocyst were similar between groups (P > 0.05), as shown in Table 1. However, the cloned blastocyst rate using donor cells treated with 100 nm TSA was higher than in the other groups (69.9 ± 4.7% v. 43.6 ± 4.3%, 43.1 ± 5.8%, or 46.6 ± 3.6%; P < 0.05), as shown in Table 1. These data suggest that proper TSA treatment for donor cells before somatic cloning improves the rate of development of porcine handmade cloned embryos to the blastocyst stage. Further research is needed to examine the in vivo development of embryos reconstructed with TSA-treated donor cells. Table 1. Developmental ability of cloned pig embryos derived fromTSA-treated donor cells

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