scholarly journals 60 HISTOLOGICAL COMPARISONS BETWEEN NUCLEAR TRANSFER AND IN VIVO PORCINE EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 180
Author(s):  
L. Overman ◽  
L. Lai ◽  
H.-T. Cheong ◽  
G.-S. Im ◽  
K.-W. Park ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Nuclear Transfer ◽  
High Rate ◽  
Normal Numbers ◽  
Diagnostic Laboratory ◽  
Invaluable Tool ◽  
Embryonic Loss ◽  
Mitotic Figures

Due to the high rate of embryonic loss during a nuclear transfer pregnancy, cloning is considered a relatively inefficient process. However, as the only method of producing knockout domestic animals it is considered an invaluable tool for the biotechnical industry. By histologically comparing embryos at significant stages in the porcine pregnancy (Days 10, 12, and 14), factors contributing to embryonic loss may be revealed. Many consider the period between days 10 and 14 to be critical for determining survivability as the embryos must undergo rapid changes to signal for maternal recognition of pregnancy as well as adapt to a changing environment. This study included three gilts per stage of pregnancy and four different experimental groups for each stage studied: nonpregnant animals, in vivo-pregnant animals, nuclear transfer (NT) recipients, and in vitro-manipulated recipients (IVM). IVM embryos were in vitro-produced embryos upon which a mock nuclear transfer has been performed in an effort to account for the variability introduced by the actual technique. Animals either were bred or underwent a surgical embryo transfer on Day 1 of the estrous cycle according to their assigned experimental group. Fifty embryos were transferred per embryo transfer. Embryos were flushed from the uterine horns at time of collection (Day 10, 12, or 14) and preserved in 10% neutral buffered formalin. All embryonic disc diameters and gross morphology were evaluated as parameters for normal development. Embryos were then dehydrated in ethanol, paraffin-imbedded, sectioned, stained with hemotoxylin and eosin, and Day 14 embryos were evaluated for abnormalities such as higher-than-normal nucleoli numbers, increased cytoplasmic vacuoles, and higher than normal numbers of mitotic figures. All results were analyzed using ANOVA. There were significant differences (P < .0001) between diameters of the embryonic disks, with the diameters of the NT embryonic disks being smaller than those of the in vivo controls at all stages studied. Morphologically, the in vivo controls were more developmentally competent than their NT counterparts by the time they reached Day 14 (P = 0.0002) in that most had achieved the more advanced elongated form of growth as opposed to remaining spherical in shape. Significant histological differences in the number of nucleoli per nuclei were also found between in vivo and NT embryos (P = 0.05) as well as between MNT and NT embryos (P = 0.05). Therefore, nuclear transfer embryos develop at a much slower rate than their in vivo counterparts and often exhibit histological abnormalities that could contribute to this slow growth. Due to the apparent increase in nucleoli, it is possible that NT embryos are being arrested at a specific stage in the cell cycle. The authors would like to acknowledge the Research Animal Diagnostic Laboratory for their help in imbedding, sectioning, and staining the embryos; Dr. Duane Keisler for running the hormone assays; and Kristin Whitworth, Melissa Samuel, Aaron Bonk, Jin-Geol Kim, Emily Fergason, David Wax, Tom Cantley, August Rieke, Randy Farrell, and Lacey Griesbaum for all their help.

scholarly journals Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos: implications for embryonic loss

Reproduction ◽  
2008 ◽  
Vol 136 (4) ◽  
pp. 433-445 ◽  
Author(s):  
Natalie I Alexopoulos ◽  
Poul Maddox-Hyttel ◽  
Pernille Tveden-Nyborg ◽  
Nancy T D'Cruz ◽  
Tayfur R Tecirlioglu ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Reproductive Technologies ◽  
Embryonic Mortality ◽  
High Rate ◽  
Morphological Examination ◽  
Embryonic Loss ◽  
The Impact

In ruminants, the greatest period of embryonic loss coincides with the period of elongation when the embryonic disc is formed and gastrulation occurs prior to implantation. The impact of early embryonic mortality is not only a major obstacle to the cattle breeding industry but also impedes the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by eitherin vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy, immunohistochemistry, and transmission electron microscopy to establishin vivodevelopmental milestones. Following morphological examination, samples were characterized for the presence of epiblast (POU5F1), mesoderm (VIM), and neuroectoderm (TUBB3). On D14, only 25, 15, and 7% of IVP, SUZI, and HMC embryos were recovered from the embryos transferred respectively, and similar low recovery rates were noted on D21, suggesting that most of the embryonic loss had already occurred by D14. A number of D14 IVP, SUZI, and HMC embryos lacked an epiblast, but presented trophectoderm and hypoblast. When the epiblast was present, POU5F1 staining was limited to this compartment in all types of embryos. At the ultrastructural level, SCNT embryos displayed abundant secondary lysosomes and vacuoles, had fewer mitochondria, polyribosomes, tight junctions, desmosomes, and tonofilaments than their IVP counterparts. The staining of VIM and TUBB3 was less distinct in SCNT embryos when compared with IVP embryos, indicating slower or compromised development. In conclusion, SCNT and to some degree, IVP embryos displayed a high rate of embryonic mortality before D14 and surviving embryos displayed reduced quality with respect to ultrastructural features and differentiation markers.

80 USE OF THE SHORT DISPOSABLE NEEDLE SYSTEM FOR FOLLICLE ASPIRATION IN MARES

2010 ◽  
Vol 22 (1) ◽  
pp. 198
Author(s):  
K. Song ◽  
W. Lee ◽  
Y. Chun ◽  
I. Lee ◽  
S. Yeon ◽  
...  
Keyword(s):  
Stainless Steel ◽  
Embryo Transfer ◽  
Recovery Rate ◽  
Nuclear Transfer ◽  
Double Lumen ◽  
Stainless Steel Needle ◽  
Disposable Needle ◽  
Follicle Aspiration

To achieve success in equine somatic cell nuclear transfer (SCNT), it is important to obtain recipient oocytes of good quality. Transvaginal ultrasoundguided follicle aspiration (TVUFA) is one of the methods to obtain recipient oocytes in equine SCNT, but the commercial long double-lumen needle for TVUFA in large animals is not currently purchasable. The aims of the present study were (1) to compare the recovery rate of short disposable needle system (14G) with that of the long double-lumen needle (12G) and (2) to investigate the developmental competency of recovered oocytes after SCNT and embryo transfer (ET). A real-time ultrasound scanner (Mylab30 vet, Esaote, Italy) equipped with a 7.5-MHz convex array transducer (model EC123) housed in a plastic vaginal device with stainless steel needle guidance was used for TVUFA from pre-ovulatory follicles 25 to 40 mm in diameter on synchronized thoroughbred mares between 6 and 9 years of age. Two types of needles were used: (1) a 12G double-lumen long needle (V-EOAD-1260L; Cook, Brisbane, Australia); (2) a 14G single-lumen disposable needle (2.1 × 80mm; Bovi-vet, Kruuse, Denmark) inserted with 18G inner needle (0.8 × 600 mm) using stainless steel connector and tube. Recovered oocytes were matured in vitro in TCM-199 with 5 mU mL-1 FSH (Folltropin-V, Bioniche, Belleville, ON, Canada) and 10% fetal bovine serum (Sigma, St. Louis, MO, USA) for 12 to 16 h. Matured oocytes were enucleated and electrically fused with equine skin fibroblasts (2.25 kV cm-1, 20μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 1 mM 6-DMAP. Immediately following SCNT procedures, cloned embryos were surgically transferred to the oviducts of recipient mares (n = 2 to 5 embryos per recipient) that had ovulated within 24 hours before the transfer. An initial pregnancy examination was performed using transrectal ultrasonography between Days 14 and 16 (Day 0 = surgery). The recovery rate of the short disposable needle (n = 89, 44.1%) was slightly increased compared with that of the long needle (n = 34, 29.8%), but the difference was not significant. Nineteen SCNT embryos were transferred to 8 mares, and 1 mare is maintaining pregnancy for 60 days. The results of this study demonstrated that our short disposable needle system could be used instead of the commercial long needle and in vivo development of oocyte recovered with the 14G needle could be maintained after nuclear transfer and embryo transfer.

ECONOMIC PERSPECTIVES OF BIOTECHNOLOGIES USING IN ANIMAL FARMING

1970 ◽  
pp. 57-60
Author(s):  
I. F. Gorlov ◽  
A. A. Mosolov ◽  
G. V. Komlatskiy ◽  
M. A. Nesterenko ◽  
K. D. Nimbona ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Economic Effect ◽  
Dairy Herd ◽  
State Level ◽  
Modern Level ◽  
Economic Perspectives ◽  
The Cost ◽  
Short Time

The article presents materials on the study of the possibility of reproduction and increase in the herd of highly productive cows through the use of embryo transplantation technology. The classical (in vivo) and more modern, developing (in vitro) methods of embryotransfer, their positive and negative sides are considered in detail. The possibility of accelerating the breeding process by using the method of transplantation, in which from one cow can be obtained from 10 to 100 calves, which will allow for 4-5 years, almost any herd (of any size and breed) with the help of biotechnology to turn into a cattle-breeding enterprise of the most modern level. At the same time, heifers obtained from unproductive cows can be used as "surrogate" mothers who are transplanted with the best donor embryos, which allows to obtain a full-fledged offspring adapted to local environmental conditions. A detailed scheme of obtaining, evaluation, storage, as well as the cost and economic effect of embryo transplantation was calculated, the market was evaluated, the required annual volume of transplants and the number of donor cows for large livestock farms were determined. As a positive example of "Scientific-production enterprise "Centre of biotechnology and embryo transfer" in 2014, implemented a project for accelerated replacement and genetic improvement of the dairy herd, engraftment averaged 57-69%, and the economic effect of the enterprise from getting a single animal by the method of embryo transfer, compared with imports of similar close in quality, ranged from 60 to 100 thousand rubles on his head. It is shown that it is necessary to organize at the state level a developed service for embryo transplantation to reduce the cost of embryo transfer and the possibility of creating in a short time in the country's own highly productive breeding nucleus of dairy and beef cattle, which will reduce, and in the future completely eliminate, import dependence on cattle products.

Effects of rate of development of in vitro-produced ovine embryos on sex ratio and in vivo survival after embryo transfer

1997 ◽  
Vol 48 (8) ◽  
pp. 1369-1378 ◽  
Author(s):  
S.L. Catt ◽  
J.K. O'Brien ◽  
W.M.C. Maxwell ◽  
G. Evans
Keyword(s):  
Sex Ratio ◽  
Embryo Transfer ◽  
Rate Of Development

scholarly journals STEM-26. SMALL MOLECULE DRUG CBL0137 INHIBITS THE FACT COMPLEX AND SENSITIZES GLIOBLASTOMA CANCER STEM CELLS TO RADIOTHERAPY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii201-ii202
Author(s):  
Miranda Tallman ◽  
Abigail Zalenski ◽  
Amanda Deighen ◽  
Morgan Schrock ◽  
Sherry Mortach ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
High Rate ◽  
Orthotopic Model ◽  
In Vivo Models ◽  
Specific Cytotoxicity ◽  
Treatment Paradigm ◽  
Cancer Agent

Abstract Glioblastoma (GBM) is a malignant brain tumor with nearly universal recurrence. GBM cancer stem cells (CSCs), a subpopulation of radio- and chemo-resistant cancer cells capable of self-renewal, contribute to the high rate of recurrence. The anti-cancer agent, CBL0137, inhibits the FACT (facilitates chromatin transcription) complex leading to cancer cell specific cytotoxicity. Here, we show that CBL0137 sensitized GBM CSCs to radiotherapy using both in vitro and in vivo models. Treatment of CBL0137 combined with radiotherapy led to increased DNA damage in GBM patient specimens and failure to resolve the damage led to decreased cell viability. Using clonogenic assays, we confirmed that CBL0137 radiosensitized the CSCs. To validate that combination therapy impacted CSCs, we used an in vivo subcutaneous model and showed a decrease in the frequency of cancer stem cells present in tumors as well as decreased tumor volume. Using an orthotopic model of GBM, we confirmed that treatment with CBL0137 followed by radiotherapy led to significantly increased survival compared to either treatment alone. Radiotherapy remains a critical component of patient care for GBM, even though there exists a resistant subpopulation. Radio-sensitizing agents, including CBL0137, pose an exciting treatment paradigm to increase the efficacy of irradiation, especially by inclusively targeting CSCs.

scholarly journals A novel phosphoramide compound, DCZ0805, shows potent anti-myeloma activity via the NF-κB pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xuejie Gao ◽  
Bo Li ◽  
Anqi Ye ◽  
Houcai Wang ◽  
Yongsheng Xie ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Burden ◽  
High Rate ◽  
Cell Counting ◽  
Tumor Xenograft ◽  
Luciferase Reporter ◽  
Therapeutic Effects ◽  
Xenograft Mouse Model

Abstract Background Multiple myeloma (MM) is a highly aggressive and incurable clonal plasma cell disease with a high rate of recurrence. Thus, the development of new therapies is urgently needed. DCZ0805, a novel compound synthesized from osalmide and pterostilbene, has few observed side effects. In the current study, we intend to investigate the therapeutic effects of DCZ0805 in MM cells and elucidate the molecular mechanism underlying its anti-myeloma activity. Methods We used the Cell Counting Kit-8 assay, immunofluorescence staining, cell cycle assessment, apoptosis assay, western blot analysis, dual-luciferase reporter assay and a tumor xenograft mouse model to investigate the effect of DCZ0805 treatment both in vivo and in vitro. Results The results showed that DCZ0805 treatment arrested the cell at the G0/G1 phase and suppressed MM cells survival by inducing apoptosis via extrinsic and intrinsic pathways. DCZ0805 suppressed the NF-κB signaling pathway activation, which may have contributed to the inhibition of cell proliferation. DCZ0805 treatment remarkably reduced the tumor burden in the immunocompromised xenograft mouse model, with no obvious toxicity observed. Conclusion The findings of this study indicate that DCZ0805 can serve as a novel therapeutic agent for the treatment of MM.

scholarly journals Inhibition of Glycolysis Suppresses Cell Proliferation and Tumor Progression In Vivo: Perspectives for Chronotherapy

2021 ◽  
Vol 22 (9) ◽  
pp. 4390
Author(s):  
Jana Horváthová ◽  
Roman Moravčík ◽  
Miroslava Matúšková ◽  
Vladimír Šišovský ◽  
Andrej Boháč ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Progression ◽  
Umbilical Vein ◽  
Cell Metabolism ◽  
High Rate ◽  
Ki 67 ◽  
Immunodeficient Mice ◽  
Inhibition Of Glycolysis

A high rate of glycolysis is considered a hallmark of tumor progression and is caused by overexpression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Therefore, we analyzed the possibility of inhibiting tumor and endothelial cell metabolism through the inhibition of PFKFB3 by a small molecule, (E)-1-(pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one (PFK15), as a promising therapy. The effects of PFK15 on cell proliferation and apoptosis were analyzed on human umbilical vein endothelial cells (HUVEC) and the human colorectal adenocarcinoma cell line DLD1 through cytotoxicity and proliferation assays, flow cytometry, and western blotting. The results showed that PFK15 inhibited the proliferation of both cell types and induced apoptosis with decreasing the Bcl-2/Bax ratio. On the basis of the results obtained from in vitro experiments, we performed a study on immunodeficient mice implanted with DLD1 cells. We found a reduced tumor mass after morning PFK15 treatment but not after evening treatment, suggesting circadian control of underlying processes. The reduction in tumor size was related to decreased expression of Ki-67, a marker of cell proliferation. We conclude that inhibition of glycolysis can represent a promising therapeutic strategy for cancer treatment and its efficiency is circadian dependent.

scholarly journals STEM-20. RADIO-SENSITIZING GLIOBLASTOMA CANCER STEM CELLS BY INHIBITION OF FACT

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi237-vi238
Author(s):  
Miranda Montgomery ◽  
Abigail Zalenski ◽  
Amanda Deighen ◽  
Sherry Mortach ◽  
Treg Grubb ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Cancer Stem Cells ◽  
Combination Therapy ◽  
High Rate ◽  
Primary Role ◽  
Cancer Cell Growth ◽  
Treatment Paradigm

Abstract Glioblastoma (GBM) has a particularly high rate of recurrence with a 5-year overall survival rate of approximately 5%. This is in part due to a sub-population of cancer stem cells (CSC), which are both radioresistant and chemotherapeutically resistant to conventional treatments. Here we investigated CBL0137, a small molecule form of curaxin, in combination with radiotherapy as a means to radiosensitize CSCs. CBL0137 sequesters FACT (facilitates chromatin transcription) complex to chromatin, which leads to activation of p53 and inhibition of NF-κB. This sequestering of FACT results in cytotoxicity especially within tumor cells and prevents FACT from performing its primary role as a histone chaperone, as well as inhibits its part in the DNA damage response pathway. We show that when combined with radiotherapy, CBL0137 administration limited the ability of CSCs to identify and repair damaged DNA. CSCs treated in vitro with CBL0137 and irradiation showed an increased inhibition of cancer cell growth and decreased viability compared to irradiation or drug alone. Combination therapy also showed more DNA damage in the CSCs than with either agent alone. Based on our in vitro evidence for the efficacy of combination therapy to target CSCs, we moved forward to test the treatment in vivo. Using a subcutaneous model, we show that the amount of CD133+ cells (a marker for GMB CSCs) was reduced in irradiation plus CBL0137 compared to either treatment alone. Survival studies demonstrated that irradiation plus CBL0137 compared to irradiation alone or CBL0137 alone increase lifespan. Here we show the ability of CBL0137, in combination with irradiation, to target patient GBM CSCs both in vitro and in vivo. This work establishes a new treatment paradigm for GBM that inclusively targets CSCs and may ultimately reduce tumor recurrence.

scholarly journals Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication

2010 ◽  
Vol 84 (19) ◽  
pp. 9864-9878 ◽  
Author(s):  
Michael E. Abram ◽  
Andrea L. Ferris ◽  
Wei Shao ◽  
W. Gregory Alvord ◽  
Stephen H. Hughes
Keyword(s):  
Viral Replication ◽  
Mutation Rate ◽  
Hot Spots ◽  
High Rate ◽  
Published Data ◽  
Single Cycle ◽  
Efficient System ◽  
Hiv 1

ABSTRACT There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZα reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 × 10−5 mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZα, the sites favored for mutations (hot spots) in lacZα depended on which strand of lacZα was present in the viral RNA. The pattern of hot spots seen in lacZα in vivo did not match any of the published data obtained when purified RT was used to copy lacZα in vitro.

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