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      Dog cloning with in vivo matured oocytes obtained using electric chemiluminescence immunoassay-predicted ovulation method

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          Abstract

          Radioactive immunoassay (RIA) is a traditional serum hormone assay method, but the application of the method in reproductive studies is limited by the associated radioactivity. The aim of present study was to evaluate the reliability of RIA and to compare its canine serum progesterone concentration determination accuracy to that of the electric chemiluminescence immunoassay (ECLI). In vivo matured oocytes were utilized for canine somatic cell nuclear transfer (SCNT), and serum progesterone levels were assessed to accurately determine ovulation and oocyte maturation. Canine serum progesterone concentrations during both proestrus and estrus were analyzed by RIA and ECLI to determine the ovulation day. Although both methods detected similar progesterone levels before ovulation, the mean progesterone concentration determined using ECLI was significantly higher than of RIA three days before ovulation. Following ovulation, oocytes were collected by surgery, and a lower percentage of mature oocytes were observed using ECLI (39%) as compared to RIA (67%) if 4-8ng/ml of progesterone were used for determination of ovulation. A high percentage of mature oocytes was observed using ECLI when 6–15 ng/mL of progesterone was used for ovulation determination. To determine whether ECLI could be used for canine cloning, six canines were selected as oocyte donors, and two puppies were obtained after SCNT and embryo transfer. In conclusion, compared to the traditional RIA method, the ECLI method is a safe and reliable method for canine cloning.

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          Most cited references 26

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          Sheep cloned by nuclear transfer from a cultured cell line.

          Nuclear transfer has been used in mammals as both a valuable tool in embryological studies and as a method for the multiplication of 'elite' embryos. Offspring have only been reported when early embryos, or embryo-derived cells during primary culture, were used as nuclear donors. Here we provide the first report, to our knowledge, of live mammalian offspring following nuclear transfer from an established cell line. Lambs were born after cells derived from sheep embryos, which had been cultured for 6 to 13 passages, were induced to quiesce by serum starvation before transfer of their nuclei into enucleated oocytes. Induction of quiescence in the donor cells may modify the donor chromatin structure to help nuclear reprogramming and allow development. This approach will provide the same powerful opportunities for analysis and modification of gene function in livestock species that are available in the mouse through the use of embryonic stem cells.
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            Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts.

            Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in sheep milk. Two cloned transfectants and a population of neomycin (G418)-resistant cells were used as donors for nuclear transfer to enucleated oocytes. Six transgenic lambs were liveborn: Three produced from cloned cells contained factor IX and neo transgenes, whereas three produced from the uncloned population contained the marker gene only. Somatic cells can therefore be subjected to genetic manipulation in vitro and produce viable animals by nuclear transfer. Production of transgenic sheep by nuclear transfer requires fewer than half the animals needed for pronuclear microinjection.
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              Eight calves cloned from somatic cells of a single adult.

              Eight calves were derived from differentiated cells of a single adult cow, five from cumulus cells and three from oviductal cells out of 10 embryos transferred to surrogate cows (80 percent success). All calves were visibly normal, but four died at or soon after birth from environmental causes, and postmortem analysis revealed no abnormality. These results show that bovine cumulus and oviductal epithelial cells of the adult have the genetic content to direct the development of newborn calves.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                13 March 2017
                2017
                : 12
                : 3
                Affiliations
                National Institute of Animal Science, RDA, Wanju, Republic of Korea
                Peking University Third Hospital, CHINA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: MZ SL.

                • Data curation: SL.

                • Formal analysis: SL.

                • Funding acquisition: T-YH.

                • Investigation: MZ YN.

                • Methodology: MZ SL.

                • Project administration: T-YH G-SI.

                • Resources: SL JN.

                • Supervision: T-YH.

                • Validation: T-YH.

                • Visualization: MZ.

                • Writing – original draft: MZ.

                • Writing – review & editing: MZ.

                Article
                PONE-D-16-43870
                10.1371/journal.pone.0173735
                5348006
                28288197
                © 2017 Lee et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 5, Tables: 1, Pages: 10
                Product
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100003627, Rural Development Administration;
                Award ID: PJ01092801
                Award Recipient :
                This work was supported by a fund of Project No. PJ01092802 from Rural Development Administration, Republic of Korea. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Germ Cells
                OVA
                Oocytes
                Medicine and Health Sciences
                Endocrinology
                Endocrine Physiology
                Menstrual Cycle
                Ovulation
                Biology and Life Sciences
                Physiology
                Endocrine Physiology
                Menstrual Cycle
                Ovulation
                Medicine and Health Sciences
                Physiology
                Endocrine Physiology
                Menstrual Cycle
                Ovulation
                Biology and Life Sciences
                Physiology
                Reproductive Physiology
                Menstrual Cycle
                Ovulation
                Medicine and Health Sciences
                Physiology
                Reproductive Physiology
                Menstrual Cycle
                Ovulation
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Amniotes
                Mammals
                Dogs
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Cloning
                Research and Analysis Methods
                Molecular Biology Techniques
                Cloning
                Biology and Life Sciences
                Biochemistry
                Hormones
                Lipid Hormones
                Progesterone
                Medicine and Health Sciences
                Women's Health
                Maternal Health
                Pregnancy
                Embryo Transfer
                Medicine and Health Sciences
                Women's Health
                Obstetrics and Gynecology
                Pregnancy
                Embryo Transfer
                Research and Analysis Methods
                Immunologic Techniques
                Immunoassays
                Medicine and Health Sciences
                Diagnostic Medicine
                Diagnostic Radiology
                Ultrasound Imaging
                Research and Analysis Methods
                Imaging Techniques
                Diagnostic Radiology
                Ultrasound Imaging
                Medicine and Health Sciences
                Radiology and Imaging
                Diagnostic Radiology
                Ultrasound Imaging
                Custom metadata
                All relevant data are within the paper and its Supporting Information files.

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