Green fluorescence was induced in the cultured PBMC of three pups after adding doxycycline to the culture media (supporting data). A total of 203 in vivo oocytes were recovered and 182 oocytes were subjected to enucleation for somatic cell nuclear transfer. To collect in vivo matured oocytes, oviducts of oocyte donors were flushed with Hepes‐buffered TCM‐199 (Invitrogen) (Lee et al.,2005). Biol Reprod. 3a) with no adverse clinical signs during the two weeks of doxycycline administration. 2016 Jun;41:1-6. doi: 10.1016/j.jevs.2016.03.008. For the controls, genomic DNA was also extracted from Tet‐on eGFP 3 (P) and a nontransgenic beagle (N). The transgenic mice have been genetically modified so that they carry a green fluorescent protein which glows green under blue light. 2008 Mar;78(3):425-31. doi: 10.1095/biolreprod.107.065185. Initially, the improvement of genetic traits was done by selective breeding methods. 1a) were established. Moderator: Dr. Nicole Pereira Pregnancy diagnosis was performed by ultrasonography at least 26 days after embryo transfer. Live Births from Domestic Dog (Canis familiaris) Embryos Produced by In Vitro Fertilization. You can change your ad preferences anytime. Three clones having transgenes were born and one dog was alive. On Day 30 after insemination, pregnancy was confirmed by ultrasonography. Cumulus cells were removed and the oocytes were enucleated by micromanipulation (Jang et al.,2007). 921 0 obj <>stream Tet‐on eGFP cells containing an eGFP reporter gene and an rtTA2S‐M2 transactivator sequence (Fig. The oxytocin system of mice and men—Similarities and discrepancies of oxytocinergic modulation in rodents and primates. Briefly, fetal fibroblasts infected with a Tet‐on eGFP vector were used for somatic cell nuclear transfer. National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error. Use the link below to share a full-text version of this article with your friends and colleagues. 3b). Germline modification of domestic animals. eCollection 2015. Furthermore, eGFP gene expression in this study showed a pattern of rapid reduction to the pretreatment level within 5 days after doxycycline removal as demonstrated by rapid reduction of fluorescence intensity in skin (Fig. To prove integration of the eGFP gene into the offspring, skin samples were collected from the pups on Day 4 after birth. A positive Southern signal was detected using a DIG luminescent detection kit (Roche). The doxycycline was dissolved in 5% sucrose to mask the bitter taste and orally administered every other day for two weeks. On Day 60 after embryo transfer, Tet‐on eGFP 3 delivered naturally four pups that weighed 140 (pup1, male), 180 (pup2, female), 180 (pup3, female), and 230 (pup4, female) g, respectively (Fig. 3b). For PCR analysis, a primer pair for the eGFP gene, upstream (5′‐CAGTGCTTCAGCCGC TACCC‐3′) and downstream (5′‐ AGTTCACCTTGATGC CGTTCTT‐3′) primers, were used to amplify a 289 bp DNA fragment. 3a) as well asin peripheral mononuclear cells (data not shown)and the eGFP band signal in Western blotting (Fig. A transgenic animal is where you take a piece of DNA that's not normally found in that animal and place it back among its normal chromosomes. If you do not receive an email within 10 minutes, your email address may not be registered, This site needs JavaScript to work properly. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP-fibroblasts. PLoS One. (b) Tet‐on eGFP cells were cultured in the absence or presence of doxycycline (Dox, 1 μg/ml) for 24 h. (c) Cells were cultured in the presence of doxycycline (1 μg/ml) for five days and then in its absence for seven days. (a) Photograph of Tet‐on eGFP 2 and 3 on Day 3 after birth. Slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. 1. Transgenic animals can be used to model human diseases. 3b). The announcement of Herman's creation caused an ethical storm. and you may need to create a new Wiley Online Library account. Shin CW, Kim GA, Park WJ, Park KY, Jeon JM, Oh HJ, Kim MJ, Lee BC. Clipboard, Search History, and several other advanced features are temporarily unavailable. - Dr. Chandini Rao However, lately dogs have lost some merit mainly due to the well‐known difficulty in manipulating genetic information (Luvoni et al.,2006; Otoi et al.,2000). Propagation of elite rescue dogs by somatic cell nuclear transfer. Nagashima JB, Sylvester SR, Nelson JL, Cheong SH, Mukai C, Lambo C, Flanders JA, Meyers-Wallen VN, Songsasen N, Travis AJ. In this study, we used a universal doxycycline‐inducible vector that contained both a driver and a target transgene (Kues et al.,2006), and an enhanced green fluorescent protein (eGFP) was used as the target gene to be expressed. Mean fluorescence intensity for the infected cells was assessed by FACS. 2b and c). 1a). Looks like you’ve clipped this slide to already. Trangenic animals are the animals with the modified genome. USA.gov. The Tet‐on eGFP cells were cultured with DMEM supplemented with 10% FBS and 1% nonessential amino acid (NEAA, Sigma‐Aldrich Corp., St. Louis, MO) in the presence of 1 μg/ml of doxycycline for five days and then cultured without the doxycycline for seven days. 2015 Dec 9;10(12):e0143930. This site needs JavaScript to work properly. While a constitutively active promoter is effective to express a target gene, the uncontrollable expression often results in unwanted outcomes. TRANSGENIC The Nonhuman Primate in Nonclinical Drug Development and Safety Assessment. 2003 Jun;68(6):2013-23. doi: 10.1095/biolreprod.102.010066. Oncomouse • 1st patented transgenic animal • Created by Philip Leder and Timothy Stewart of Harvard University in 1984. Transgenic plants can be made by introducing foreign DNA into a variety of different tissues. %%EOF doi: 10.1371/journal.pone.0143930. endstream endobj startxref So for example, you can make a transgenic by having a piece of DNA that you clone in a laboratory and inject it into a fertilized egg of a mouse embryo, for example, then that becomes integrated into the chromosome. In short, after lysis of the specimens, supernatants were collected and loaded onto a 12% SDS‐polyacrylamide gel. Expansion of the tetracycline-dependent regulation toolbox for Helicobacter pylori. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. All dogs were cared for in separate indoor facilities and managed following a standard procedure established by the Committee for Accreditation of Laboratory Animal Care (approval number: SNU‐090508‐5) and according to the Guideline for the Care and Use of Laboratory Animals of Seoul National University. The kinetics of eGFP expression in the Tet‐on system show a very tight regulation dependent on doxycycline as the expression of the transgene was progressively increased and maintained at a high level and decreased by doxycycline treatment (Szulc et al.,2006). A cloned beagle named Ruppy – short for Ruby Puppy – is the world’s first transgenic dog. 1b) and by FACS analysis revealed a tight regulation of the transgene by doxycycline (Fig. If you continue browsing the site, you agree to the use of cookies on this website. The results of Western blot analysis also showed a robust eGFP band signal which appeared after doxycycline administration (Fig. When the female cloned dog reached puberty, serum progesterone concentration was monitored for detecting timing of ovulation (Hong et al.,2009b). V�����k�Eb�q�e-eug�dmc�d�`�a�f���e���WiU���5|K2�dž33���d�ʙΨ���1\�%�8�SCΙ����O8��a�g`�� ��@���0� fN``����3�iI�GP>� -����/8`�s��([[E���Y�|�"�C��|(���S�� � �7 I have absolutely no negative side effects. The virus‐containing medium harvested from the cell culture was filtered and used to infect female beagle fetal fibroblasts. Duration of gestation in pregnant dogs carrying cloned fetuses. Generation of cloned transgenic cats expressing red fluorescence protein. (a) Four puppies were delivered from Tet‐on eGFP 3. Learn about our remote access options, Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, 151‐744, Korea, Department of Physiology, Catholic University of Daegu School of Medicine, Daegu, Korea, Teoan Kim, Department of Physiology, Catholic University of Daegu School of Medicine, Daegu, Korea, Byeong Chun Lee, Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, 151‐744, Korea, Stem Cell, RNL BIO Ltd., Seoul, 153‐768, Korea, Department of Clinical Sciences, University of Kentucky, Lexington, Kentucky, USA. Considering the potential toxicity that may be caused by this high dose of doxycycline in rodents (Holst and Phemister,1971), it is of interest to determine if lower doses than the currently used ones or the dose that we administered in this study would be sufficient to induce genes of interest in other species. I usually order a research paper or English essay here and there. The induced transgene expression detected by epifluorescence microscopy (Fig. The Transgenic Animal Service of Queensland (TASQ) is a division of The University of Queensland Biological Resources (UQBR) department. As of this date, Scribd will manage your SlideShare account and any content you may have on SlideShare, and Scribd's General Terms of Use and Privacy Policy will apply. Number of times cited according to CrossRef: The Domestic Dog Embryo: In Vitro Fertilization, Culture, and Transfer.

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