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transgenic sheep production

Cas9 and sgRNA plasmids were constructed per the previously published method (Han et al., 2014). It simplifies the introduction process, prevents the spread of disease and provides technology to establish animal embryo libraries. Boland, M. (1984) Use of the rabbit oviduct as a screening tool for the viability of mam. Hence, the OPS vitrification approach on microinjected embryos may be a valuable method of ET to avoid wasting embryos and to synchronize recipients, thus reducing unnecessary animal sacrifices (Zhao et al., 2013).

In humans, pregnancy rates for frozen pronuclear embryos were 45.5%, and the offspring survival rate was 20.9% (Wong & Wong, 2011). Reprod Fertil Dev. Moor, R. M. and Trounson, A. O. This may relate to regulative effects of melatonin on reproductive hormone production. Third World Congress Genet. For example, AANAT transgenic tomatoes and rice exhibited significantly higher melatonin levels than did their wild-types (Okazaki et al., 2009; Park et al., 2013; Kang et al., 2010; Wang et al., 2014). The acquired pronuclear embryos were immediately microinjected and cultured in vitro at 38.5 °C in a chamber with 5% CO2 and a humidified atmosphere within 30 min. of survival offspring/No. This service is more advanced with JavaScript available, Transgenesis Techniques In addition, the mean birth weight of the offspring was significantly higher in the frozen group than that in the non-frozen group (4.4 kg vs. 3.6 kg, P < 0.05), this was consistent with an observation of human neonates (Vergouw et al., 2012). Yingjie Wu contributed reagents/materials/analysis tools. The transgenic gene was analyzed by Genome PCR.

The OPS vitrification approach may be a valuable method in microinjected- embryo transfer technology, which could reserve embryos and result in fewer unnecessary animal sacrifices. After the IVT, the sgRNA and Cas9-encoding mRNA were purified by ethanol and lithium chloride separately, then precipitated and dissolved in RNase-free water, and finally stored at −80 °C until use. (1989) Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice. Melatonin and other reagents, unless specified, were purchased from Sigma Chemical Co. (Sigma Aldrich, St. Louis, MO, USA). The production of transgenic sheep has proven difficult compared to the mouse and lower animals. Another important observation in the current study was that the reproductive functions, including superovulation, pregnancy rate and average number of surviving lambs in the transgenic offspring with overexpression of AANAT were improved compared to their non-transgenic counterparts. (2013). Polymorphism in the ovine keratin-associated protein gene KRTAP7-1 and its association with wool characteristics. Data are expressed as means ± SEM and were analyzed by AVOVA followed by Student’s t-test using GraphPad Prism (San Diego, CA, USA). The intact AANAT CDS was amplified, and the Xho I restriction site was added (634 bp full-length). In 1997, the open pulled straw (OPS) method was reported. These differences were species-specific as the litter size order is mice > sheep > humans. 7-Phloroeckol promotes hair growth on human follicles in vitro. The corrected cDNA was verified by Sanger sequencing, excised from the T-vector by Xho I, and subsequently cloned into the mammary gland-specific expression vector, pBC1 (Invitrogen, Inc., Carlsbad, CA, USA). Different superscripts in the same column differ significantly (P < 0.05). There were no significant differences in fiber diameter, medullation, and hogget body weight. The open pulled straw (OPS) vitrification method has been successfully applied in mouse, pig, and goat embryos as well as in buffalo oocytes, but it has not yet been applied to the microinjected embryos. Naunyn Schmiedebergs Arch Pharmacol. Superovulation in the donors was induced by injecting 320 IU of PMSG (Ningbo Hormone Products CO., Ltd, Ningbo, Zhengjiang, China) 12 h after sponge removal. Learn more, Over 10 million scientific documents at your fingertips. This process is experimental and the keywords may be updated as the learning algorithm improves. Ewe estrous cycles were synchronized by a 14-day treatment of progesterone with a controlled internal drug release (CIDR) device containing 300 mg progesterone (EAZI-BREED® CIDR® Sheep and Goats Device Pfizer Animal Health, New Zealand). of average lamb survival: no. of offspring/No. From the 146th day of pregnancy, ewes were kept indoors (two ewes/pen) to monitor the lambing initiation. All transgenic sheep production and sample collection procedures strictly followed the protocols approved by the Animal Welfare Committee of China Agricultural University (Permission Number: SYXK [Beijing] 2015002; the period of valid days: September 22, 2015–September 22, 2020), and the study was conducted in strict accordance with the guidelines and regulations established by this committee. Epub 2017 Aug 10. LH production was similar to FSH production in both groups. Few reports were on the influence of OPS vitrification and thawing on microinjected pronuclear embryo quality in mammals, particularly in ovine. Sixty-three donors were used to produce non-frozen microinjected pronuclear embryos. Survival rates in mouse pronuclear embryos subjected to OPS freezing were shown to be significantly lower than those of the morulae (Zhou et al., 2005). Notably, she is the first transgenic farm mammal ever created.

The AANAT gene coding sequence (CDS) was amplified using TransStart FastPfu DNA Polymerase (TransGen Biotech, Beijing, China). The mixture of mRNA from Cas9/sgRNA and DNA from AANAT was injected into the pronuclear embryo cytoplasm.

Furthermore, melatonin also significantly elevates serum estradiol-17β (E2) content, which may be mediated by MT2 activation (Jia et al., 2016). 2018;14(11):1850-1869. doi: 10.1080/15548627.2018.1490852. However, the benefits to human and animal health and agricultural productivity are potentially enormous (Ward and Nancarrow, Chapter 5) and support for the continuation of the work is assured. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. After thawing, 122 embryos were viable and the survival rate was 89.1% (122/137). After ET, recipient pregnancies were detected by ultrasonography on the 60th day. The production of transgenic sheep has proven difficult compared to the mouse and lower animals. Tian X, Lv D, Ma T, Deng S, Yang M, Song Y, Zhang X, Zhang J, Fu J, Lian Z, Zhu S, Wu Y, Xing Y, Liu G. (A) Microinjected embryos before vitrification; (B) microinjected embryos after thawing. Ward, K. A., Murray, J. D., Shanahan, C. M., Rigby, N. W., and Nancarrow, C. D. (1988) The creation of transgenic sheep for increased wool productrvity, in, Kanagawa, H., Abas-Mazni, O., and Valdez, C. A. AANAT transgenic sheep generated via OPS vitrified-microinjected pronuclear embryos and reproduction efficiency of the transgenic offspring https://t.co/e7TWxy8BPO https://t.co/1p6RXvok3u, AANAT transgenic sheep generated via OPS vitrified-microinjected pronuclear embryos and reproduction efficiency of the transgenic offspring https://t.co/19POYvNpnk @thePeerJ https://t.co/NB23IPGVWE. Finally, they were picked up by the narrow end of the OPS within 25 s and immediately plunged into liquid nitrogen.

Naunyn Schmiedebergs Arch Pharmacol. Gagne, M. B., Pothier, F., and Sirard, M.-A. 70.32.89.170. (1991) Expression and physiology of performance regulating genes in transgenic sheep. Yukun Song conceived and designed the experiments, performed the experiments, approved the final draft, embryo transfer. Baicalin, a flavonoid, affects the activity of human dermal papilla cells and promotes anagen induction in mice. 1996 Feb;14(2):181-4. doi: 10.1038/nbt0296-181. Total RNA was extracted from the pineal gland of the White Suffolk using Trizol Reagent (Invitrogen Inc., Carlsbad, CA, USA) per the manufacturer’s instructions. Pregnancy was diagnosed by identifying at least one fetal image. New polymorphism in the 5' flanking region of. These data may provide references for human-assisted reproduction. "Following" is like subscribing to any updates related to a publication. Transgenic chickens are now able to synthesize human proteins in the "white" of their eggs.

Miller, B. G. and Moore, N. W. (1976) Effect of progesterone and oestradiol on RNA and protein metabolism in the genital tract and on survival of embryos in the ovariectomised ewe.

Melatonin has potent protective effects on embryo quality, especially under cold stress (Jang et al., 2010).

Products were sequenced by Sangon Biotech, Beijing, and blasted in the GenBank database. These keywords were added by machine and not by the authors. Nancarrow, C. D., Marshall, J. T. A., Clarkson, J. D., Murray, J. D., Millard, R. M., Shanahan, C. M., Wynn, P. C., and Ward, K. A. Gandolfi, R, Brevini, T. A. L., and Moor, A. M. (1989) Effect of oviduct environment on embryonic development.

 |  2014 Aug;387(8):789-93. doi: 10.1007/s00210-014-0986-0.

Embryo transfer efficiencies of frozen/non-frozen microinjected pronuclear embryos and the positive rate of the offspring.

thank you in advance for your patience and understanding. The recombinant vectors were referred to as pBC1-AANAT. We Perez, A., Solano, R., Castro, F. O., Leonart, R. L., de Armas, R., Martinez, R., Aguilar, A., Herrara, L., and de la Fuente, J. Five transgenic lambs resulted from the microinjection of 591 embryos; one male and one female showed IGF1 expression in the skin. The authors would like to thank Pro. Shin SH, Bak SS, Kim MK, Sung YK, Kim JC. The transgenic-positive rates were 63.5% and 60% in the non-frozen and frozen groups, respectively.

Our promise Springer Nature is developing a new tool to find and evaluate Protocols. The following information was supplied regarding data availability: The raw data are provided in a Supplemental File. A total of 1,007 embryos were collected, and 855 were microinjected and transplanted into 167 recipients after 30–60 min of the microinjection, yielding a utilization rate of 98.5% (855/868).

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