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Cloning Animals by Somatic Cell Nuclear Transfer--Biological Factors

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Cloning Animals by Somatic Cell Nuclear Transfer--Biological Factors University of Connecticut OpenCommons@UConn ANSC Articles Department of Animal Science November 2003 Cloning Animals by Somatic Cell Nuclear Transfer- -Biological Factors X Cindy Tian Chikara Kubota Brian Enright Xiangzhong Yang Follow this and additional works at: https://opencommons.uconn.edu/ansc_articles Recommended Citation Tian, X Cindy; Kubota, Chikara; Enright, Brian; and Yang, Xiangzhong, "Cloning Animals by Somatic Cell Nuclear Transfer-- Biological Factors" (2003). ANSC Articles. 5. https://opencommons.uconn.edu/ansc_articles/5 Reproductive Biology and Endocrinology BioMed Central Review Open Access Cloning animals by somatic cell nuclear transfer – biological factors X Cindy Tian1, Chikara Kubota2, Brian Enright1 and Xiangzhong Yang*1,3 Address: 1Center for Regenerative Biology/Department of Animal Science, University of Connecticut, Storrs, Connecticut 06269-4243, USA, 2Kagoshima Prefectural Cattle Breeding and Genetic Institute, Kagoshima, Japan and 3Evergen Biotechnologies, Inc. Incubator Program at the University of Connecticut, 1392 Storrs Road, Storrs, CT06269-USA Email: X Cindy Tian - [email protected]; Chikara Kubota - [email protected]; Brian Enright - [email protected]; Xiangzhong Yang* - [email protected] * Corresponding author Published: 13 November 2003 Received: 08 August 2003 Accepted: 13 November 2003 Reproductive Biology and Endocrinology 2003, 1:98 This article is available from: http://www.rbej.com/content/1/1/98 © 2003 Tian et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. nuclear transferdonor cell typesdonor ageserum starvationcell passage Abstract Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other specie, this review will be focused on somatic cell cloning of cattle. Introduction potential for therapeutic cloning and allo-transplantation Somatic cell cloning (cloning or nuclear transfer) is a tech- [6]. In addition to its practical applications, cloning has nique in which the nucleus (DNA) of a somatic cell is become an essential tool for studying gene function [7], transferred into an enucleated metaphase-II oocyte for the genomic imprinting [8], genomic re-programming [9– generation of a new individual, genetically identical to the 12], regulation of development, genetic diseases, and gene somatic cell donor (Figure 1). The success of cloning an therapy, as well as many other topics. entire animal, Dolly, from a differentiated adult mam- mary epithelial cell [1] has created a revolution in science. One of the most difficult challenges faced, however, is It demonstrated that genes inactivated during tissue differ- cloning's low efficiency and high incidence of develop- entiation can be completely re-activated by a process mental abnormalities [13–19]. Currently, the efficiency called nuclear reprogramming: the reversion of a differen- for nuclear transfer is between 0–10%, i.e., 0–10 live tiated nucleus back to a totipotent status. Somatic cloning births after transfer of 100 cloned embryos. Developmen- may be used to generate multiple copies of genetically tal defects, including abnormalities in cloned fetuses and elite farm animals, to produce transgenic animals for placentas, in addition to high rates of pregnancy loss and pharmaceutical protein production or xeno-transplanta- neonatal death have been encountered by every research tion [2–5], or to preserve endangered species. With opti- team studying somatic cloning. It has been proposed that mization, it also promises enormous biomedical Page 1 of 7 (page number not for citation purposes) Reproductive Biology and Endocrinology 2003, 1 http://www.rbej.com/content/1/1/98 b). Donor cell culture a). Donor animal h). Transfer to recipients i). Clone birth c). Matured d). Enucleation g). Cloned embryo e). Nuclear transfer oocytes f). Cell fusion development SchematicFigure 1 diagram of the somatic cloning process Schematic diagram of the somatic cloning process. Cells are collected from donor (a) and cultured in vitro (b). A matured oocyte (c) is then enucleated (d) and a donor cell is transferred into the enucleated oocyte (e). The somatic cell and the oocyte is then fused (f) and the embryos is allowed to develop to a blastocyst in vitro (g). The blastocyst can then be transferred to a recipient (h) and cloned animals are born after completion of gestation (i). low cloning efficiency may be largely attributed to the cuss several strategies used to improve nuclear transfer incomplete reprogramming of epigenetic signals [20–23]. efficiencies. Factors affecting nuclear reprogramming Serum starvation of donor cells Various strategies have been employed to modify donor Serum starvation was used in the creation of Dolly and cells and the nuclear transfer procedure in attempts to was believed essential to the success of nuclear transfer improve the efficiency of nuclear transfer. Most of these [1]. Serum starvation induces quiescence of cultured cells, efforts are focused on donor cells. These include: a) syn- and arrests them at the cell cycle stage of G0. Most labora- chrony of the cell cycle stage of donor cells [24–26], as tories that have succeeded with nuclear transfer have uti- well as synchrony between donor cells and recipient lized a serum starvation treatment. However, there is a oocytes [27,28]; b) using somatic cells from donors of var- debate as to whether inducing quiescence is required for ious ages [29–33], tissue origins [26,34–39], passages successful nuclear transfer. Cibelli et al. [52] proposed [16,40,41] and culture conditions [42]; c) transfer of stem that G0 was unnecessary and that calves could be pro- cells with low levels of epigenetic marks [43–48]; and d) duced from cycling cells. In his study, actively dividing modifying epigenetic marks of donor cells with drugs bovine fibroblasts were used for nuclear transfer and four [49–51]. Although the efficiency of nuclear transfer has calves were born from 28 embryos transferred to 11 recip- been dramatically improved from the initial success rate ients. Because 56% of cycling cells in that study were in G1 of one live clone born from 277 embryo transfers [1], stage, it is likely that all cloned animals produced in this none of the aforementioned efforts abolished the com- study were from donor cells at G1 stage. Cells at G2, S or mon problems associated with nuclear transfer. These M would not be expected to generate cloned animals in observations suggest that further studies on nuclear repro- this study because they are incompatible with the recipi- gramming are needed in order to understand the underly- ent oocytes used. This study demonstrated that cells at G1 ing mechanisms of reprogramming and significantly stage can produce live cloned animals and G0 induction improve the ability of the differentiated somatic nuclei to is not essential. be reprogrammed. In the following section, we will dis- Page 2 of 7 (page number not for citation purposes) Reproductive Biology and Endocrinology 2003, 1 http://www.rbej.com/content/1/1/98 Table 1: Development of embryos cloned from donor cells from were obtained from embryos derived from the "shake-off" a 17-year old bull with and without serum starvation treatment cells, whereas no embryos from the confluent cells sur- Serum No. No (%) No (%) No (%) vived beyond 180 days of gestation. Kasinathan et al. [25] starvation oocytes fused cleaved blastocysts concluded that nuclear transfer donor cell cycle stage is important, particularly effecting late fetal development, Yes 288 114 (40) 75 (66) 24 (21)a and that actively dividing G1 cells support higher devel- No 282 102 (36) 79 (78) 28 (28)a opment rates than cells in G0. Despite the fact that Kasi- nathan's study did not produce live clones from G0 cells, Values with the same superscripts are not significantly different (P > a high nuclear transfer success rate was obtained by Cho 0.05). et al. [55] who subjected donor cells to serum starvation and found no improvement in blastocyst development from adult donor cells, but resulted in a 27.3% calving Since the report of Cibelli and colleagues, many laborato- rate. ries have compared nuclear transfer using donor cells with and without serum starvation. In our study, we used cells To further complicate the matter, Wells et al. [26] com- from a 17-year old male Japanese Black beef bull and pared two different types of non-transfected bovine fetal found that serum starvation was not required for success- fibroblasts (BFFs) that were synchronized in G0, G1 or ful cloning because cloned embryos and animals were different phases within G1. They showed that serum star- produced from cells not subjected
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