23 PRODUCTION OF HANDMADE CLONED GOAT BLASTOCYSTS USING FETAL FIBROBLAST CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 91 ◽  
Author(s):  
Y. S. Akshey ◽  
D. Malakar ◽  
A. K. De
Keyword(s):  
Uv Light ◽  
Light Exposure ◽  
Research Work ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
Uv Illumination ◽  
Fetal Fibroblasts ◽  
Cone Formation ◽  
Handmade Cloning

Nuclear transfer is a very effective method for propagation of desired, extinct, and endangered animals as well as for the production of 100% transgenic animals. Enucleated oocytes and somatic cells are required for nuclear cloning. For enucleation, DNA-specific stains are used for visualization of the metaphase (MII) plate in matured oocytes under UV illumination in both micromanipulator-based and handmade cloning techniques. The present study was carried out to produce cloned goat embryos using the handmade cloning approach. Fetal fibroblast cells were used as nuclear donors (passages 3–4). Oocytes were collected from slaughterhouse-derived ovaries and matured in maturation medium (TCM-199 (HEPES modified), 5 µg mL–1 FSH, 10 µg mL–1 LH, 1 µg mL–1 estradiol-17β, 50 µg mL–1 gentamicin, 3 mg mL–1 BSA, and 10% inactivated estrus goat serum) at 38.5�C in 5% CO2 in air with maximum humidity for 24 h. We observed that the formation of transparent protrusion cones on the surface of the in vitro-matured goat oocytes was clearly visible under the stereomicroscope after zona digestion with 2 mg mL–1 pronase. The extent of protrusion cone formation in matured oocytes was 95–100% within 20–30 min in handling medium T 20 (TCM-199 + 20% FCS). The MII plate in the protrusion cone was confirmed (100%) after Hoechst 33342 staining and subsequent UV illumination under the inverted microscope. Zona-free oocytes were bisected on the basis of the protrusion cone by a microblade in medium (T 20 + 2.5 µg mL–1 cytochalasin B) for enucleation. Enucleated demi-oocytes were selected which had no protrusion cone and were without staining. Fetal fibroblasts from confluent monolayers were used. Two demi-oocytes were coupled with one trypsinized fetal fibroblast cell using 200 µg mL–1 phytohemagglutinin. The triplets were fused together with a combination of alternating current (7 V) and direct current (2.31 kV cm–1 for 15 µs with a double pulse) in fusion medium (0.3 m mannitol, 0.1 mM MgSO4, 0.05 mm CaCl2, and 3 mg mL–1 BSA). Four h after fusion, reconstructed oocytes were activated by using 2 µm Ca Ionophore for 5 min at room temperature and incubated with 2 mm 6-dimethylaminopurine at 38.5�C in 5% CO2 in air for 3 h. Activated reconstructed embryos were cultured in embryo development medium (TCM-199, 10% FCS, essential and nonessential amino acids, and 10 mg mL–1 BSA) in the well of the well (WOW) culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 258–264) at 38.5�C in 5% CO2 in air. In the present study, fusion, cleavage, and morula and blastocyst formation rates were 180/200 (90%), 72/180 (40%), 56/72 (77%), and 6/56 (11%), respectively. Further studies will be required to optimize blastocyst production. In conclusion, the protrusion cone formation in matured goat oocytes made it convenient for bisection and enucleation without Hoechst staining and UV light exposure, enabling the production of goats from handmade somatic cell cloning. The Council of Scientific and Industrial Research, India, has provided a fellowship to the first author to carry out this research work.

scholarly journals 75HIGHLY EFFICIENT AND RELIABLE CHEMICALLY ASSISTED ENUCLEATION METHOD FOR HANDMADE CLONING IN CATTLE AND SWINE

2004 ◽  
Vol 16 (2) ◽  
pp. 159 ◽  
Author(s):  
G. Vajta ◽  
T.T. Peura ◽  
L. Lai ◽  
C.N. Murphy ◽  
R.S. Prather ◽  
...  
Keyword(s):  
Uv Light ◽  
Polar Body ◽  
Cumulus Cells ◽  
Dependent Manner ◽  
Uv Illumination ◽  
Reconstructed Embryo ◽  
Fetal Fibroblasts ◽  
Membrane Protrusions ◽  
Handmade Cloning

In bovine and porcine nuclear transfer, most traditional enucleation procedures require potentially harmful chromatin staining and UV illumination. The purpose of our work was to find an efficient and reliable chemically-assisted procedure for enucleation connected to the handmade cloning (HMC) technique without chromatin staining. Slaughterhouse-derived oocytes were collected and matured in vitro. At 21 (bovine) or 43 (porcine) h after the start of maturation, cumulus cells were removed with vortexing and oocytes were further incubated in the maturation medium supplemented with 0.5μgmL−1 demecolcine for 2h. Subsequently, zonae pellucidae were digested with 2mgmL−1 pronase in the presence of 10% cattle serum (CS) for 6 to 8min and washed in HEPES-buffered TCM-199 medium and 20% CS. Bisection was performed in the same medium by hand under a stereomicroscope by using a microblade. A small membrane protrusion observable on the surface of oocytes was used as an orientation point. One-third of the cytoplasm connected to this protrusion was removed, and the cytoplasts and karyoplasts were collected separately. Bovine cytoplasts were used as recipients for HMC experiments (Vajta et al., 2003, Biol. Reprod. 68, 571–578) with fetal fibroblasts as donors, and reconstructed embryos were cultured for 7 days. In Experiment 1 (3 replicates), the possibility of oriented bisection at different time points was determined on a total of 225 bovine oocytes. At 5, 15, 25, 35 and 55min after the end of pronase digestion 64, 91, 93, 72 and 59% of oocytes had membrane protrusions (P<0.05 between all groups, SAS Genmod) illustrating the time-dependent manner of the protrusion. In Experiment 2, the efficiency and reliability of enucleation was measured. Bisection was performed between 5 and 35min after pronase digestion. Subsequently both supposed cytoplasts and karyoplasts were stained with Hoechst and investigated under UV light. In cattle (9 replicates), bisection was successfully performed in 94% (519/552) of oocytes, and 98% (507/517) of those bisected were enucleated, i.e. the chromatin was entirely in the presumptive karyoplast. In swine (3 replicates), 91% (302/331) of oocytes were successfully bisected and 95% (280/296) were enucleated. In Experiment 3 (cattle; 4 replicates), blastocyst per reconstructed embryo rates were 47% (139/293), illustrating the high developmental ability in vitro. Considering that no oocyte selection based on the presence of polar body was performed, the above system seems to be more efficient and reliable than other enucleation methods. Moreover, expensive equipment (inverted fluorescent microscope) and a potentially harmful step (staining and UV illumination) can be eliminated from the HMC without compromising the high in vitro efficiency.

scholarly journals Influence of co-culture during maturation on the developmental potential of equine oocytes fertilized by intracytoplasmic sperm injection (ICSI)

Reproduction ◽  
2001 ◽  
pp. 925-932 ◽  
Author(s):  
X Li ◽  
LH Morris ◽  
WR Allen
Keyword(s):  
Epithelial Cells ◽  
Intracytoplasmic Sperm Injection ◽  
Fibroblast Cells ◽  
Cell Stage ◽  
Developmental Potential ◽  
Fetal Fibroblast ◽  
Nuclear Maturation ◽  
Fetal Fibroblasts ◽  
Oviduct Epithelial Cells

The influence of co-culture with either oviduct epithelial cells or fetal fibroblast cells on in vitro maturation of equine oocytes and their potential for development to blastocysts and fetuses after intracytoplasmic sperm injection (ICSI) was investigated. The oocytes were obtained from ovaries from abattoirs and were matured in vitro for 28-30 h in TCM-199 only, or in TCM-199 co-culture with oviduct epithelial cells or fetal fibroblast cells. Metaphase II oocytes were subjected to ICSI with an ionomycin-treated spermatozoon. The injected oocytes were cultured for 7-9 days in Dulbecco's modified Eagle's medium. Morphologically normal early blastocysts were transferred to the uteri of recipient mares. Nuclear maturation rates and the rates of cleavage to the two-cell stage for injected oocytes were similar in the groups of oocytes that were matured in TCM-199 (49 and 63%), in co-culture with oviduct epithelial cells (53 and 65%) or in co-culture with fetal fibroblasts (51 and 57%). There were no significant differences in the proportions of blastocysts that developed from the two-cell embryos derived from oocytes matured by co-culture with either oviduct epithelial cells (30%) or fetal fibroblasts (17%). However, significantly higher proportions of blastocysts were produced from both these co-culture groups than from the groups of oocytes matured in TCM-199 only (P < 0.05). Six of the blastocysts that had developed from oocytes co-cultured with oviduct epithelial cells were transferred into recipient mares and four pregnancies resulted. These results demonstrate a beneficial influence of co-culture with either oviduct epithelial cells or fetal fibroblasts for maturation of oocytes in vitro.

54 CHEMICAL ENUCLEATION OF IN VITRO-MATURED BUFFALO (BUBALUS BUBALIS) OOCYTES INDUCED BY DEMECOLCINE FOR HANDMADE SOMATIC CELL CLONING

2008 ◽  
Vol 20 (1) ◽  
pp. 108
Author(s):  
R. A. Shah ◽  
M. S. Chauhan ◽  
R. S. Manik ◽  
S. K. Singla
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Uv Light ◽  
Genetic Material ◽  
Bubalus Bubalis ◽  
Significant Loss ◽  
Control Group ◽  
Cone Formation ◽  
Handmade Cloning

Cloning by somatic cell nuclear transfer requires enucleation of the recipient oocyte to remove its genetic material. In handmade cloning, a simplified cloning procedure (Vajta et al. 2001 Cloning 3, 89–95), zona-free oocytes are manually bisected, stained, and exposed to UV light for selection of demi-oocytes devoid of chromosomes. Development of procedures for enucleation which avoid exposure to UV light and conserve most of the cytoplasmic volume are necessary for improving the efficiency of handmade cloning. Chemically assisted enucleation protocols involve treating cumulus–oocyte complexes (COCs) during IVM with cytoskeleton-modifying agents, like demecolcine, which induce protrusion cone formation on the surface of the oocytes (Tani et al. 2006 Cloning Stem Cells 8, 61–66). Such a cone-like structure can then be easily excised with a microblade and enucleation of the oocyte achieved without a significant loss of cytoplasm. The aim of the present study was to establish an efficient protocol for demecolcine treatment of buffalo COCs during IVM to obtain the maximal proportion of oocytes where chromosomes are either expelled into the surface protrusion cone or completely enucleated. In Experiment I, COCs (n = 244), obtained from slaughterhouse buffalo ovaries and matured in vitro in TCM-199 (containing 10% FBS, 5 µg mL–1 pFSH, and 0.81 mm sodium pyruvate) at 38.5�C (in 5% CO2, 90–95% relative humidity), were treated in two groups with demecolcine (0.5 µg mL–1) beginning at either 15 h or 19 h from the start of IVM up to the end of IVM at 22 h, and compared to a control group (without demecolcine). Data were analyzed using ANOVA. The proportion of oocytes where a protrusion cone was observed was greatest (P < 0.05) in the 15-h treatment group (84%, n = 72/86) compared to those in either the 19-h or control groups (58%, n = 46/78, and 60%, n = 48/80, respectively). The presence of demecolcine for the last 7 h of IVM appears to have a significant effect on protrusion cone formation in buffalo oocytes. In Experiment II, COCs (n = 276)were divided into four groups, matured in vitro this time for 24 h and treated with demecolcine (0.5 µg mL–1) from the onset of IVM and up to 18, 21, or 24 h, and compared to a control group without demecolcine. Protrusion cone formation was observed in 72% (52/72), 66% (40/60), 62% (40/64), and 70% (56/80) of oocytes, respectively, in these groups. These percentages did not differ significantly (P < 0.05). However, the 21-h treatment resulted in complete enucleation in 32% (20/64) of oocytes, which was significantly greater (P < 0.05) than that in the other three groups where no such enucleation was observed. It can be hypothesized that demecolcine-free treatment from 21 to 24 h may have assisted in inducing complete enucleation of a significant number of treated oocytes. In conclusion, these results show that demecolcine-assisted and induced enucleation procedures can be used for increasing the efficiency of oocyte enucleation in handmade cloning and other nuclear transfer procedures in buffalo.

83 IN VITRO DEVELOPMENTAL ABILITIES OF PORCINE CLONED EMBRYOS RECONSTITUTED WITH CELL NUCLEI OF GENETICALLY TRANSFORMED FETAL FIBROBLASTS CYTOMETRICALLY DIAGNOSED ON CELL CYCLE AND APOPTOSIS

2007 ◽  
Vol 19 (1) ◽  
pp. 159
Author(s):  
M. Samiec ◽  
M. Skrzyszowska ◽  
M. Bochenek ◽  
D. Lipinski ◽  
R. Slomski
Keyword(s):  
Cell Cycle ◽  
High Efficiency ◽  
Fibroblast Cells ◽  
Apoptotic Cells ◽  
Facs Analysis ◽  
Developmental Potential ◽  
Fetal Fibroblast ◽  
Donor Cells ◽  
Fetal Fibroblasts

The important factor that determines the development of mammalian cloned embryos is structuro-functional quality of nuclear donor cells. Analysis of nuclear DNA (nDNA) content of somatic cells undergoing apoptosis has become one of the most common methods for single-parameter flow cytometric measurement of this process. Apoptosis assessment is performed by quantification of hypodiploid cells. The aim of our study was to examine the in vitro developmental potential of porcine nuclear transfer (NT) embryos reconstituted with non-apoptotic fetal fibroblast cells expressing the eGFP transgene. The nuclear donor cells were derived from cell line populations whose representative random samples had been analyzed on both cell cycle and apoptosis through non-vital nDNA fluorescent dyeing and flow cytometry (FACS). Frozen-thawed fibroblast cells, which had been cultured up to a total confluency after 2–4 passages, were used for the diagnostics. The cells were fixed in ice-cold 70% ethanol. Then, the fetal fibroblasts were exposed to nDNA extraction buffer for 5 min at room temperature, and incubated in DNA staining solution (propidium iodide and RNAse) for 30 min. After fluorescent labeling, the cells were analyzed in the flow cytometer by reading nDNA fluorescence in the red band. In vitro-matured oocytes were the source of recipient cells. 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. FACS analysis revealed that, out of all of the diagnosed fetal fibroblast cells, 54.7% were cycling, and up to 45.3% were late-apoptotic. In turn, from among the normal (i.e. non-apoptotic) cells, 82.2% were at G0/G1 stages of cell cycle, 17.0% at the S stage, and 0.8% at G2/M stages. A total of 150 enucleated oocytes were successfully fused with non-apoptotic transgenic nuclear donor cells. Out of 150 cultured NT embryos, 123 (82.0%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages yielded 53/150 (35.3%) and 37/150 (24.7%), respectively. In conclusion, the FACS analysis for mitotic cycle of 100%-confluent transgenic fetal fibroblasts confirmed the high efficiency of the cell cycle synchronization at G0/G1 phases. However, a contact inhibition method induced the high frequency of late-apoptotic cells. Moreover, the relatively high percentage of NT blastocysts was developed from oocytes reconstructed with eGFP transgenic fetal fibroblast cells. 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 34 LIFESPAN AND CHROMOSOMAL STABILITY OF BOVINE AND PORCINE FETAL FIBROBLAST CELLS CULTURED IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 167 ◽  
Author(s):  
A.M. Giraldo ◽  
J.W. Lynn ◽  
C.E. Pope ◽  
R.A. Godke ◽  
K.R. Bondioli
Keyword(s):  
Cell Lines ◽  
Cultured Cells ◽  
Culture Conditions ◽  
Cycle Duration ◽  
Fibroblast Cells ◽  
Proliferative Capacity ◽  
Chromosomal Stability ◽  
Fetal Fibroblasts ◽  
Fetal Bovine

The low efficiency of nuclear transfer (NT) has been related to factors such as mitochondria heteroplasmy, failure of genomic activation, and asynchrony between the donor karyoplast and recipient cytoplast. Few studies have characterized donor cell lines in terms of proliferative capacity and chromosomal stability. It is known that suboptimal culture conditions can induce chromosomal abnormalities, and the use of aneuploid donor cells during NT can lead to a high incidence of abnormal cloned embryos (Giraldo et al. 2004 Reprod. Fertil. Dev. 16, 124 abst). The purpose of this study was to determine the lifespan and chromosomal stability of bovine and porcine fetal cells. Four bovine and four porcine fibroblast cells lines were established from 50-day and 40-day fetuses, respectively. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C in 5% CO2. Each cell line was passaged to senescence. Total population doublings (PDs) and cell cycle duration were calculated. To determine the chromosome numbers at different PDs, cells were synchronized in metaphase, fixed, and stained. ANOVA and chi-square tests were used to analyze differences in PDs and proportion of aneuploid cells between cell lines, respectively (P < 0.05). The results show that proliferative capacity was not different between cell lines derived from the same species. Cell lines derived from bovine and porcine fetuses had different in vitro lifespans (33 PDs vs. 42 PDs, respectively; P < 0.05). The mean length of the cell cycles for both bovine and porcine fetal fibroblasts was ∼28 h. The percentage of aneupliod cells in both bovine and porcine fetal cell lines increased progressively with duration of culture (see Table) and was high throughout the study. The proliferative capacity of cultured cells was similar within individuals of the same species, but growth characteristics differed between fetal bovine and porcine cell lines. The progressive increase of aneuploid cells could be due to suboptimal culture conditions or unusual chromosome instability in the particular fetuses used. These data demonstrate the importance of determining chromosome content and the use of cells at early passages to decrease the percentage of aneuploid reconstructed embryos and increase the efficiency of NT.

29 EVALUATION OF DIFFERENT FUSION PARAMETERS IN THE RECONSTRUCTION OF SWINE HANDMADE CLONING EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 95
Author(s):  
C. Feltrin ◽  
A. S. Lima ◽  
M. Monaco ◽  
S. M. Wilson ◽  
D. Kim ◽  
...  
Keyword(s):  
Uv Light ◽  
Polar Body ◽  
Donor Cell ◽  
Fusion Rate ◽  
Blastocyst Stage ◽  
Cleavage Rate ◽  
Reconstructed Embryos ◽  
Cloning Technique ◽  
Handmade Cloning

The goal of this experiment was to compare different fusion parameters in the handmade cloning technique to produce cloned swine embryos. After in vitro maturation of 618 oocytes, 431 (69.8%) presented a visible polar body and were used in the experiment. The next step was the removal of the cumulus oophorus cells and the digestion of the zona pellucida using pronase (5 mg mL–1) in HEPES TCM199. Oocytes were then exposed to a medium containing cytochalasin B (5 µg mL–1) for 15 min before being bisected with a hand-held blade. The bisected oocytes (cytoplasts) were then placed in medium supplemented with Hoechst 33342 and exposed to UV light to select cytoplasts without metaphase II plates. Next, two cytoplasts and a mesenchymal stem cell (nucleus donor) were pushed together in a phytohemagglutinin (550 µg mL–1) solution. Once adhered, these structures were divided into 3 groups (G) to be fused using different parameters: (G1) 2 pulses (DC) of 0.6 kV cm–1 for 30 µs, (G2) 2 pulses (DC) of 0.9 kV cm–1 for 30 µs, and (G3) 2 pulses (DC) of 1.2 kV cm–1 for 30 µs. For all three groups, 0.3 m of mannitol solution (without calcium) was used in the fusion chamber, and an initial pre-pulse (AC) of 10V for 15 s was performed to permit the alignment of 100% of the cytoplast-donor cell structures. After fusion, reconstructed embryos were activated in 0.3 m mannitol and 0.1 mm calcium in the fusion chamber using 2 pulses of 0.9 kV cm–1 for 30 µs followed by incubation in 10 µg mL–1 of cycloheximide solution for 4 h. Afterwards, the reconstructed embryos were transferred to NCSU23 medium supplemented with amino acids (nonessential and essential) and 0.4% bovine serum albumin. The embryos were cultured at 39�C in a 100% humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. Cleavage rates were evaluated after 48 h of culture. For G1, the fusion rate was 43% (25/58) with 72% cleavage (18/25), the G2 fusion rate was 87% (56/64) with 80% cleavage (45/56), and the G3 fusion rate was 79% (53/67) with 69% cleavage (37/53). Statistical analysis was performed using the chi-square test. There were no significant differences in fusion rates between groups G2 and G3, but the fusion rate of these groups was significantly different from that of G1 (P < 0.05). No significant differences in cleavage rate were observed among the three groups. In conclusion, fusion using 2 pulses at either 0.9 or 1.2 kV cm–1 for 30 µs was more efficient for embryo reconstruction in the handmade cloning technique compared to that using 2 pulses at 0.6 kV cm–1 for 30 µs. Further studies need to be performed to improve cleavage rates and assess development to the blastocyst stage.

61 EFFICIENCY OF TWO ENUCLEATION METHODS FOR THE PRODUCTION OF TRANSGENIC PIG EMBRYOS BY HANDMADE CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 148 ◽  
Author(s):  
J. Li ◽  
Y.-H. Zhang ◽  
Y.-T. Du ◽  
P. M. Kragh ◽  
S. Purup ◽  
...  
Keyword(s):  
High Efficiency ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Transgenic Pig ◽  
Transgenic Pigs ◽  
Uv Illumination ◽  
Polar Bodies ◽  
Transgenic Embryos ◽  
Handmade Cloning

Since the successful production of transgenic pigs by somatic nuclear transfer (Lai et al. 2002 Science 295, 1089–1092), more efficient reproduction technologies for transgenic pigs have been in demand. The purpose of our work was to develop an efficient method for production of transgenic embryos by handmade cloning (HMC; Vajta et al. 2001 Cloning 3, 89–95) connected to oriented enucleation to eliminate potential harm of staining and UV illumination at cytoplast selection. After 41–42 h of in vitro maturation, oocytes were further cultured with or without 0.4 µg mL−1 demecolcine for 45 min (i.e. chemically assisted handmade enucleation (CAHE) vs. oriented handmade enucleation (OHE)). Subsequently, the cumulus cells were removed and zonae pellucidae were partially digested. Oocytes with visible extrusion cones or polar bodies attached to the surface were subjected to oriented bisection. The putative cytoplasts without extrusion cones or polar bodies, containing the major part of cytoplasm, were selected as the recipients. Two cytoplasts were electro-fused with one transgenic fibroblast expressing either amyloid precursor protein (APP) or green fluorescent protein (GFP), while non-transgenic fibroblasts were used as control nuclear donors. After activation (Kragh et al. 2005 Theriogenology 64, 1536–1545; Du et al. 2005 Cloning Stem Cells 7, 199–205), reconstructed embryos were cultured in porcine zygote medium-3 for 7 days. The rates of cleavage and blastocyst cell numbers were recorded on Day 2 and Day 7, respectively. In 5 replicates, the correct bisection efficiency achieved with CAHE was higher compared to that with the OHE method (93 ± 1% vs. 82 ± 2%, respectively; P &lt; 0.05). Table 1 shows that blastocyst rates with APP and GFP transgenic fibroblasts as nuclear donors after CAHE were lower (P &lt; 0.05) compared to those with the OHE method; in contrast, cleavage rates of embryos from different fibroblast donors were similar and so were blastocyst rates of non-transgenic donors after either CAHE or OHE. Our results show that embryos reconstructed from APP and GFP transgenic donors have compromised in vitro developmental rates after CAHE rather than after the OHE method; however, a high efficiency with both enucleation methods was observed when using non-transgenic somatic cells. Table 1.Comparison of two enucleation methods for the production of transgenic pig embryos

scholarly journals Ferroelectric Induced UV Light-Responsive Memory Devices with Low Dark Current

Electronics ◽  
2021 ◽  
Vol 10 (16) ◽  
pp. 1897
Author(s):  
Hanleem Lee ◽  
Young Tea Chun
Keyword(s):  
Vinylidene Fluoride ◽  
Uv Light ◽  
Light Exposure ◽  
Incident Light ◽  
Colour Change ◽  
Memory Device ◽  
Interface Layer ◽  
Uv Illumination ◽  
Poly Vinylidene Fluoride ◽  
Stable Performance

We developed solution-processed hybrid photodetectors with a poly (9-vinylcarbazole)/zinc oxide nanoparticle photoactive layer and a poly (vinylidene fluoride-co-trifluoroethylene) ferroelectric copolymer buffer layer on flexible plastic substrates. The presence of a ferroelectric-poling interface layer significantly enhanced the charge transfer and responsivity of the photodetectors under ultraviolet (UV, 365 nm) light exposure. The responsivity of the device reached 250 mA/W at a reverse bias of 5 V and incident light intensity of 27.5 μW/cm2. This responsivity was four times higher than that of a device without the ferroelectric copolymer layer (64 mA/W) under the same conditions. The response time of the device to incident UV light also improved from 322 to 34 ms with the addition of the ferroelectric copolymer layer. In addition, the flexible device exhibited a stable performance in an air environment up to a maximum strain of 0.3 under bending stress. Finally, a UV-light-responsive memory device was successfully fabricated by using the developed hybrid photodetector and liquid crystals. This device showed a colour change from white to black upon UV illumination, and the on-state of the device was maintained for 30 s without light exposure owing to the polarization of poly (vinylidene fluoride-co-trifluoroethylene).

70 FETAL FIBROBLAST CELLS PERMEABILIZED WITH STREPTOLYSIN O IMPROVE THE RATES OF FUSION AND IN VITRO DEVELOPMENT OF PORCINE RECONSTRUCTED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 152
Author(s):  
K. Naruse ◽  
Y. M. Shin ◽  
Y. S. Quan ◽  
C. S. Park ◽  
D. I. Jin
Keyword(s):  
Cell Number ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
In Vitro Development ◽  
Reconstructed Embryos ◽  
Streptolysin O ◽  
Donor Cells ◽  
Developmental Rates ◽  
Vitro Development

Streptolysin O (SLO) is known to bacterial proteins that form very large pores in the plasma membrane of mammalian cells. SLO has been used in the delivery of proteins into living cells following permeabilization. The objective of this study was to investigate the effect of permeabilization of donor cells using SLO on in vitro development of porcine reconstructed embryos. Porcine fetal fibroblast cells were treated with Ca2+-free DMEM medium containing 200 ng mL−1 of SLO for 50 min before or after trypsinization. Those SLO-treated donor cells were injected into enucleated oocytes, fused with 2 DC pulses (1.2 kV cm−1, 30 µs) and cultured in procine zygote medium-3 (PZM-3) for 6 days. In vitro development of the reconstructed embryos was examined. SLO treatment after trypsinzation significantly increased (P &lt; 0.05) the percentage of fusion rates and blastocyst developmental rates compared with that before trypsinization or in the nontreated group. Additionally there were no significant differences in fusion rates, cleavage rates, blastocyst developmental rates, and total cell number of blastocysts between the SLO-treated group before trypsinzation and the nontreated group. Next, after the trypsinzation treatment, fetal fibroblast cells were incubated in Ca2+-free DMEM containing 200 ng mL−1 of SLO for 0, 30, 50, and 70 min and SLO-treated donor cells were also tested for fusion rate and developmental capability following reconstruction. The 50-min group of SLO-treated cells significantly increased (P &lt; 0.05) the percentage of fusion rates (90.6 vs. 77.6, 85.4, and 78.5%) and blastocyst developmental rates (24.7 vs. 13.5, 11.2, and 13.5%) compared with the other groups (Table 1). However, there was no significant difference in the total cell number of blastocysts among SLO-treated groups. Although cleavage rates the in SLO-treated groups were not significantly different from those of the nontreated group, there the cleavage rates were slightly in SLO-treated groups. In conclusion, permeabilization of porcine fetal fibroblast cells with SLO improves the fusion rates and in vitro development of porcine reconstructed embryos. Table 1.Effects of SLO treatment of fetal fibroblasts by different exposure times on in vitro development of porcine reconstructed embryos

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