Novel method for demonstrating nuclear contribution in mouse nuclear transfer

1998 ◽  
Vol 10 (8) ◽  
pp. 633 ◽  
Author(s):  
Megan Munsie ◽  
Teija Peura ◽  
Anna Michalska ◽  
Alan Trounson ◽  
Peter Mountford
Keyword(s):  
Transgenic Mice ◽  
Nuclear Transfer ◽  
Staining Pattern ◽  
Embryonic Cells ◽  
Simple Method ◽  
Tissue Samples ◽  
Developmental Potential ◽  
Novel Method ◽  
Nuclear Contribution ◽  
Serial Nuclear Transfer

Confirmation of nuclear contribution is essential to all nuclear transfer experiments. Contribution is easily demonstrated in nuclear transfer progeny but more difficult to confirm in nuclear transfer embryos. The use of donor nuclei isolated from lacZ transgenic mice offers a clear and simple method to demonstrate contribution in nuclear transfer embryos and offspring. The unique line of transgenic mice (Zin40) used in this study displays nuclear localised lacZ expression in all cells, including embryonic blastomeres, and demonstrates distinctive blue nuclei when treated with X-gal substrate. This characteristic staining pattern provided an ideal marker for demonstrating nuclear contribution. Nuclear transfer embryos were generated following serial nuclear transfer of metaphase-arrested nuclei from transgenic and non-transgenic 4-cell embryos. Totipotency of nuclear transfer blastocysts was confirmed by the generation of live born offspring. Transgenic blastocysts and all tissue samples from fetuses and pups generated by nuclear transfer displayed distinctive blue nuclei when stained with X-gal. This staining pattern was characteristic of the transgenic mice from which the donor nuclei were isolated and clearly confirmed nuclear origin. The use of this marker will also allow the opportunity to investigate the developmental potential of nuclear transfer embryos by examining the contribution of nuclear transfer embryonic cells in chimaeric embryos.

Modifications of chemically induced-enucleated nuclear transfer technique by reverse-order nuclear transfer in mouse

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 261-268 ◽  
Author(s):  
Yongsheng Wang ◽  
Jun Liu ◽  
Shuang Tang ◽  
Zhixing An ◽  
Zhilin Guo ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Developmental Rate ◽  
Cell Number ◽  
Reverse Order ◽  
Cleavage Rate ◽  
Simple Method ◽  
Developmental Potential ◽  
Chemically Induced ◽  
Second Polar Body

SummaryTo improve the developmental potential of somatic cell cloned embryos derived from demecolcine (DC) induced-enucleated nuclear transfer (INT), we modified the INT procedures by transferring donor nuclei into recipient cytoplasts prior to the induced enucleation of the recipient cytoplasts, and we called this modified INT technique as reverse-order and induced-enucleated nuclear transfer (RINT). Standard nuclear transfer (SNT) and INT were performed as controls. The dynamic changes of maternal and transferred donor nuclei in the RINT oocytes were monitored to evaluate the feasibility of this new nuclear transfer (NT) technique by timed immunofluorescence. Timed immunofluorescence showed that RINT is feasible because none of the transferred donor nuclei were expelled with the second polar body (Pb) in the RINT oocytes, while 42.2% of the oocytes showed extrusion of all maternal chromosome and spindles with the second Pb at 60 min after activation and DC treatment. Although there was no difference in cleavage rate (86.6% vs. 82.1%), the rates of successful enucleation and blastocyst formation were significantly increased in RINT compared with INT (44.1% vs. 27.5% and 43.3% vs. 12.8%, respectively; p < 0.01). Compared with SNT, there was no difference in cleavage rate (86.6% vs. 78.4%), but the blastocyst developmental rate was significantly increased in the RINT group (43.3% vs. 25.3%; p < 0.01). Blastocysts derived from RINT had a higher total cell number than those from SNT (45.1 ± 3 vs. 37.6 ± 4; p < 0.05). Our results provide evidence that RINT is feasible and may provide a more efficient and simple method for NT than INT.

scholarly journals Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 219-230 ◽  
Author(s):  
Feikun Yang ◽  
Ru Hao ◽  
Barbara Kessler ◽  
Gottfried Brem ◽  
Eckhard Wolf ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Epigenetic Mark ◽  
Cell Type ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Donor Cell Type

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres fromin vivofertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF,P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that inin vivofertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres fromin vivoderived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

A Novel Method for Somatic Cell Nuclear Transfer to Mouse Embryonic Stem Cells

2005 ◽  
Vol 7 (4) ◽  
pp. 265-271 ◽  
Author(s):  
Danièle Pralong ◽  
Krzysztof Mrozik ◽  
Filomena Occhiodoro ◽  
Nishanthi Wijesundara ◽  
Huseyin Sumer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Novel Method

scholarly journals A NOVEL SIMPLE-BASED PRESSURE-ENTHALPY COUPLING SCHEME FOR ENGINE FLOW PROBLEMS

2012 ◽  
Vol 17 (1) ◽  
pp. 1-20 ◽  
Author(s):  
Maximilian Emans ◽  
Zoran Žunič ◽  
Branislav Basara ◽  
Sergey Frolov
Keyword(s):  
Linear Equations ◽  
Computing Time ◽  
Simple Algorithm ◽  
Variable Density ◽  
Coupling Scheme ◽  
The Novel ◽  
Simple Method ◽  
Flow Problems ◽  
Novel Method ◽  
Coupling Terms

A novel method in CFD derived from the SIMPLE algorithm is presented. Instead of solving the linear equations for each variable and the pressurecorrection equation separately in a so-called segregated manner, it relies on the solution of a linear system that comprises the discretisation of enthalpy and pressurecorrection equation which are linked through physical coupling terms. These coupling terms reflect a more accurate approximation of the density update with respect to thermodynamics (compared to standard SIMPLE method). We show that the novel method is a reasonable extension of existing CFD techniques for variable density flows based on SIMPLE. The novel method leads to a reduction of the number of iterations of SIMPLE which translates in many – but not in all – cases to a reduction in computing time. We will therefore demonstrate practical advantages and restrictions in terms of computational efficiency for industrial CFD applications in the field of piston engine simulations.

scholarly journals Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3111
Author(s):  
Po-Yu Lin ◽  
Denny Yang ◽  
Chi-Hsuan Chuang ◽  
Hsuan Lin ◽  
Wei-Ju Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Developmental Potential ◽  
Functional Features ◽  
Early Mouse ◽  
Extraembryonic Tissues

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are ‘true’ totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely ‘cluster 3’, as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.

scholarly journals 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P&lt;0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P&lt;0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P&lt;0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

Real-time velocity scaling and obstacle avoidance for industrial robots using fuzzy dynamic movement primitives and virtual impedances

2018 ◽  
Vol 45 (1) ◽  
pp. 110-126 ◽  
Author(s):  
Iman Kardan ◽  
Alireza Akbarzadeh ◽  
Ali Mousavi Mohammadi
Keyword(s):  
Real Time ◽  
Obstacle Avoidance ◽  
Time Constant ◽  
Fuzzy System ◽  
Industrial Robots ◽  
Simple Method ◽  
Content Type ◽  
Dynamic Movement ◽  
Steering Mechanism ◽  
Novel Method

Purpose This paper aims to increase the safety of the robots’ operation by developing a novel method for real-time implementation of velocity scaling and obstacle avoidance as the two widely accepted safety increasing concepts. Design/methodology/approach A fuzzy version of dynamic movement primitive (DMP) framework is proposed as a real-time trajectory generator with imbedded velocity scaling capability. Time constant of the DMP system is determined by a fuzzy system which makes decisions based on the distance from obstacle to the robot’s workspace and its velocity projection toward the workspace. Moreover, a combination of the DMP framework with a human-like steering mechanism and a novel configuration of virtual impedances is proposed for real-time obstacle avoidance. Findings The results confirm the effectiveness of the proposed method in real-time implementation of the velocity scaling and obstacle avoidance concepts in different cases of single and multiple stationary obstacles as well as moving obstacles. Practical implications As the provided experiments indicate, the proposed method can effectively increase the real-time safety of the robots’ operations. This is achieved by developing a simple method with low computational loads. Originality/value This paper proposes a novel method for real-time implementation of velocity scaling and obstacle avoidance concepts. This method eliminates the need for modification of original DMP formulation. The velocity scaling concept is implemented by using a fuzzy system to adjust the DMP’s time constant. Furthermore, the novel impedance configuration makes it possible to obtain a non-oscillatory convergence to the desired path, in all degrees of freedom.

P–516 Evidence for self-correction in preimplantation embryos by comparative molecular karyotyping of blastocoele fluid, trophectoderm and inner cell mass

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
D Zhigalina ◽  
N Skryabin ◽  
O Kanbekova ◽  
V Artyukhova ◽  
A Svetlakov ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Morphological Characteristics ◽  
Preimplantation Embryos ◽  
Molecular Karyotyping ◽  
Embryonic Cells ◽  
Developmental Potential ◽  
Positive Correlation ◽  
Registration Number ◽  
Molecular Karyotype

Abstract Study question Does the molecular karyotype of the cell-free DNA (cfDNA) from the blastocyst fluid (BF) can predict the efficiency of self-correction of karyotype of preimplantation embryo? Summary answer Detection of aneuploidies in the BF potentially can point out on effective self-correction of blastocyst karyotype and consequently on high developmental potential of mosaic embryos. What is known already Correction of aneuploidies in the preimplantation embryos can be provided by several mechanisms, including apoptosis. The predominant death of aneuploid cells was demonstrated in mouse embryos (Bolton, 2016). A positive correlation was also shown between the concentration of cfDNA from the BF of human blastocyst and the morphology of the embryo, as well as between the activity of caspase–3 and the concentration of cfDNA (Rule, 2018). The incidence of failed amplification after WGA being significantly higher among euploid blastocysts (Magli, 2019). The capacity of abnormal cells extruding into the BF would be related to the embryo development potential (Gianaroli, 2019). Study design, size, duration This is a prospective observational study of thirty-one Day 5 human blastocysts. Cryopreserved blastocysts were received after treatment cycles at the IVF Center with informed consent obtained from couples. The average age of 15 women was 32.25±5 years. The morphological characteristics of blastocysts were estimated in accordance with the Gardner classification (Gardner, Schoolcraft, 1999). The procedure of BF aspiration and trophectoderm (TE) and ICM cells separation of the blastocysts was previously described (Tsuiko, 2018). Participants/materials, setting, methods WGA was performed by PicoPLEX kit (Rubicon Genomics, USA) or REPLI-g Mini kit (Qiagen) according to manufacturer’s protocols. The DNA of the BF, ICM and TE were analyzed separately using cCGH, aCGH and NGS. SurePrint G3 Human CGH Microarrays (8x60K, Agilent Technologies) were used according to the manufacturer’s recommendations. Image analysis was done using ISIS (v.5.5) (Metasystems) and Agilent CytoGenomics Software (v.3). VeriSeq™ PGS Kit - MiSeq® System (Illumina) was used for NGS. Main results and the role of chance Molecular karyotypes of all three samples - BF, ICM and TE, were obtained for 23 (74.2%) blastocysts. A correlation between the woman’s age and the number of aneuploidies in cfDNA (p = 0.0009) was found. A positive correlation may indicate that the number of aneuploidies in the embryonic cells increases with the age of a woman, however, the embryonic karyotype undergoes self-correcting through the elimination of aneuploid cells. It was noted that well-developing blastocysts (groups 4–5, according to Gardner’s classification) had fewer aneuploidies in ICM (p = 0.0141) and TE (p = 0.0436). In contrast, there was a tendency to an increase in the number of aneuploidies in the BF during blastocysts transition from stage 3 to 5 (p = 0.3542). We assessed the relationship between the number of aneuploidies in groups of blastocysts with different characteristics of ICM (groups “A” and “B” according to Gardner’s classification). These groups significantly differ in the number of aneuploidies in cfDNA (p = 0.0352), although the statistically significant differences between the number of aneuploidies in ICM (p = 0.5992) and in TE (p = 0.5934) was not detected. Thus, higher-quality embryos in terms of ICM morphology contain more abnormalities in the BF, since in this group the elimination of aneuploid cells is more efficient. Limitations, reasons for caution The number of embryos is limited in this study. More comprehensive studies are required to confirm the observed tendency. Wider implications of the findings: Aneuploid cells elimination can be a cause of increasing cfDNA concentration in the BF, which may be a marker of the viability of mosaic embryos when it is necessary to decide on mosaic embryo transfer. This study was supported by the RFBR (15–04–08265) and by the RSF (20–74–00064). Trial registration number Not applicable

Design of Fazl-Tash novel method for sustainable resilient comprehensive supplier selection problem

Kybernetes ◽  
2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Hamed Fazlollahtabar ◽  
Navid Kazemitash
Keyword(s):  
Supplier Selection ◽  
Huge Number ◽  
Simple Method ◽  
Content Type ◽  
Novel Method ◽  
Rank And Select ◽  
The Cost ◽  
Supplier Selection Problem ◽  
Industrial Company

Purpose However, due to the huge number of studies and on the other hand to be new and creative, the represented models and methods – as the two main parts of this field – have been got more complicated, which consequently have been turned into unpractical research studies for the realistic situations. Therefore, the purpose of this study is the representation of a novel and simple method to deal with the aforementioned gap. Design/methodology/approach To this end, Fazl-Tash method have been proposed, in which a thorough and complete model including 114 criteria and a simple technique to rank and select the best supplier have been presented. Sustainability and resiliency are considered in collecting criteria effective on supplier selection. Findings The method was carried out in a case study in an industrial company. The efficiency of the proposed method is evaluated in comparison with other conventional approaches. Originality/value As selecting the supplier plays a crucial role to bring some important advantages for companies, such as coping with the cost and time problems and influencing the majority of contemporary markets’ requirements, in recent years, there have been representing more effective studies in the supplier selection literature.

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