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Cell cycle synchronization of canine ear fibroblasts for somatic cell nuclear transfer

  • Ok Jae Koo (a1), Mohammad Shamim Hossein (a1), So Gun Hong (a1), Jose A. Martinez-Conejero (a2) and Byeong Chun Lee (a3)...


Cycle synchronization of donor cells in the G0/G1 stage is a crucial step for successful somatic cell nuclear transfer. In the present report, we evaluated the effects of contact inhibition, serum starvation and the reagents – dimethyl sulphoxide (DMSO), roscovitine and cycloheximide (CHX) – on synchronization of canine fibroblasts at the G0/G1 stage. Ear fibroblast cells were collected from a beagle dog, placed into culture and used for analysis at passages three to eight. The population doubling time was 36.5 h. The proportion of G0/G1 cells was significantly increased by contact inhibition (77.1%) as compared with cycling cells (70.1%); however, extending the duration of culture did not induce further synchronization. After 24 h of serum starvation, cells were effectively synchronized at G0/G1 (77.1%). Although synchronization was further increased gradually after 24 h and even showed significant difference after 72 h (82.8%) of starvation, the proportion of dead cells also significantly increased after 24 h. The percentage of cells at the G0/G1 phase was increased (as compared with controls) after 72 h treatment with DMSO (76.1%) and after 48 h treatment with CHX (73.0%) or roscovitine (72.5%). However, the rate of cell death was increased after 24 and 72 h of treatment with DMSO and CHX, respectively. Thus, we recommend the use of roscovitine for cell cycle synchronization of canine ear fibroblasts as a preparatory step for SCNT.


Corresponding author

All correspondence to: Byeong Chun Lee. Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151–742, Korea. Tel: +822 880 1269. Fax: +822 873 1269. e-mail:


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Boquest, A.C., Day, B.N. & Prather, R.S. (1999). Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells. Biol. Reprod. 60, 1013–9.
Campbell, K.H., Loi, P., Otaegui, P.J. & Wilmut, I. (1996). Cell cycle co-ordination in embryo cloning by nuclear transfer. Rev. Reprod. 1, 40–6.
Cho, J.K., Lee, B.C., Park, J.I., Lim, J.M., Shin, S.J., Kim, K.Y., Lee, B.D. & Hwang, W.S. (2002). Development of bovine oocytes reconstructed with different donor somatic cells with or without serum starvation. Theriogenology 57, 1819–28.
Cibelli, J.B., Stice, S.L., Golueke, P.J., Kane, J.J., Jerry, J., Blackwell, C., Ponce de Leon, F.A. & Robl, J.M. (1998). Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280, 1256–8.
Gibbons, J., Arat, S., Rzucidlo, J., Miyoshi, K., Waltenburg, R., Respess, D., Venable, A. & Stice, S. (2002). Enhanced survivability of cloned calves derived from roscovitine-treated adult somatic cells. Biol. Reprod. 66, 895900.
Goissis, M.D., Caetano, H.V., Marques, M.G., de Barros, F.R., Feitosa, W.B., Milazzotto, M.P., Binelli, M., Assumpcao, M.E. & Visintin, J.A. (2007). Effects of serum deprivation and cycloheximide on cell cycle of low and high passage porcine fetal fibroblasts. Reprod. Domest. Anim. 42, 660–3.
Gomez, M.C., Jenkins, J.A., Giraldo, A., Harris, R.F., King, A., Dresser, B.L. & Pope, C.E. (2003). Nuclear transfer of synchronized African wild cat somatic cells into enucleated domestic cat oocytes. Biol. Reprod. 69, 1032–41.
Hashem, M.A., Bhandari, D.P., Kang, S.K., Lee, B.C. & Suk, H.W. (2006). Cell cycle analysis of in vitro cultured goral (Naemorhedus claudatus) adult skin fibroblasts. Cell. Biol.Int. 30, 698703.
Hashem, M.A., Bhandari, D.P., Kang, S.K. & Lee, B.C. (2007). Cell cycle analysis and interspecies nuclear transfer of in vitro cultured skin fibroblasts of the Siberian tiger (Panthera tigris Altaica). Mol. Reprod. Dev. 74, 403–11.
Hill, J.R., Roussel, A.J., Cibelli, J.B., Edwards, J.F., Hooper, N.L., Miller, M.W., Thompson, J.A., Looney, C.R., Westhusin, M.E., Robl, J.M. & Stice, S.L. (1999). Clinical and pathologic features of cloned transgenic calves and fetuses (13 case studies). Theriogenology 51, 1451–65.
Hinrichs, K., Choi, Y.H., Love, C.C., Chung, Y.G. & Varner, D.D. (2006). Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract. Reproduction 131, 1063–72.
Hinrichs, K., Choi, Y.H., Varner, D.D. & Hartman, D.L. (2007). Production of cloned horse foals using roscovitine-treated donor cells and activation with sperm extract and/or ionomycin. Reproduction 134, 319–25.
Holker, M., Petersen, B., Hassel, P., Kues, W.A., Lemme, E., Lucas-Hahn, A. & Niemann, H. (2005). Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos. Cloning Stem Cells 7, 3544.
Hossein, M.S., Kim, M.K., Jang, G., Fibrianto, H.Y., Oh, H.J., Kim, H.J., Kang, S.K. & Lee, B.C. (2007). Influence of season and parity on the recovery of in vivo canine oocytes by flushing fallopian tubes. Anim. Reprod. Sci. 99, 330–41.
Iyer, V.R., Eisen, M.B., Ross, D.T., Schuler, G., Moore, T., Lee, J.C., Trent, J.M., Staudt, L.M., Hudson, J., Jr., Boguski, M.S., Lashkari, D., Shalon, D., Botstein, D. & Brown, P.O. (1999). The transcriptional program in the response of human fibroblasts to serum. Science 283, 83–7.
Jang, G., Kim, M.K., Oh, H.J., Hossein, M.S., Fibrianto, Y.H., Hong, S.G., Park, J.E., Kim, J.J., Kim, H.J., Kang, S.K., Kim, D.Y. & Lee, B.C. (2007). Birth of viable female dogs produced by somatic cell nuclear transfer. Theriogenology 67, 941–7.
Jang, G., Hong, S.G., Oh, H.J., Kim, M.K., Park, J.E., Kim, H.J., Kim, D.Y. & Lee, B.C. (2008a). A cloned toy poodle produced from somatic cells derived from an aged female dog. Theriogenology 69, 556–63.
Jang, G., Oh, H.J., Kim, M.K., Fibrianto, Y.H., Hossein, M.S., Kim, H.J., Kim, J.J., Hong, S.G., Park, J.E., Kang, S.K. & Lee, B.C. (2008b). Improvement of canine somatic cell nuclear transfer procedure. Theriogenology 69, 146–54.
Jiang, H., Lin, J., Su, Z.Z., Collart, F.R., Huberman, E. & Fisher, P.B. (1994). Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21, WAF1/CIP1, expression in the absence of p53. Oncogene 9, 3397–406.
Johnson, D.G. & Walker, C.L. (1999). Cyclins and cell cycle checkpoints. Annu. Rev. Pharmacol. Toxicol. 39, 295312.
Kasinathan, P., Knott, J.G., Wang, Z., Jerry, D.J. & Robl, J.M. (2001). Production of calves from G1 fibroblasts. Nat. Biotechnol. 19, 1176–8.
Kato, Y., Tani, T., Sotomaru, Y., Kurokawa, K., Kato, J., Doguchi, H., Yasue, H. & Tsunoda, Y. (1998). Eight calves cloned from somatic cells of a single adult. Science 282, 2095–8.
Khammanit, R., Chantakru, S., Kitiyanant, Y. & Saikhun, J. (2008). Effect of serum starvation and chemical inhibitors on cell cycle synchronization of canine dermal fibroblasts. Theriogenology 70, 2734.
Kues, W.A., Anger, M., Carnwath, J.W., Paul, D., Motlik, J. & Niemann, H. (2000). Cell cycle synchronization of porcine fetal fibroblasts: effects of serum deprivation and reversible cell cycle inhibitors. Biol. Reprod. 62, 412–9.
Lee, B.C., Kim, M.K., Jang, G., Oh, H.J., Yuda, F., Kim, H.J., Hossein, M.S., Kim, J.J., Kang, S.K., Schatten, G. & Hwang, W.S. (2005a). Dogs cloned from adult somatic cells. Nature 436, 641.
Lee, G.S., Kim, H.S., Hyun, S.H., Lee, S.H., Jeon, H.Y., Nam, D.H., Jeong, Y.W., Kim, S., Kim, J.H., Han, J.Y., Ahn, C., Kang, S.K., Lee, B.C. & Hwang, W.S. (2005b). Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein. Theriogenology 63, 973–91.
Okuda, A. & Kimura, G. (1988). Non-specific elongation of cell cycle phases by cycloheximide in rat 3Y1 cells, and specific reduction of G1 phase elongation by simian virus 40 large T antigen. J. Cell Sci. 91, 295302.
Onishi, A., Iwamoto, M., Akita, T., Mikawa, S., Takeda, K., Awata, T., Hanada, H. & Perry, A.C. (2000). Pig cloning by microinjection of fetal fibroblast nuclei. Science 289, 1188–90.
Pennisi, E. (2000). Profits from precious pets. Science 288, 1726.
Pines, J. (1995). Cyclins and cyclin-dependent kinases: a biochemical view. Biochem. J. 308, 697711.
Ponzio, G., Loubat, A., Rochet, N., Turchi, L., Rezzonico, R., Farahi Far, D., Dulic, V. & Rossi, B. (1998). Early G1 growth arrest of hybridoma B cells by DMSO involves cyclin D2 inhibition and p21[CIP1] induction. Oncogene 17, 1159–66.
Sherr, C.J. & Roberts, J.M. (1995). Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 9, 1149–63.
Sun, X., Wang, S., Zhang, Y., Wang, H., Wang, L., Ying, L., Li, R. & Li, N. (2008). Cell-cycle synchronization of fibroblasts derived from transgenic cloned cattle ear skin: effects of serum starvation, roscovitine and contact inhibition. Zygote 16, 111–6.
Suzuki, E., Nagata, D., Yoshizumi, M., Kakoki, M., Goto, A., Omata, M. & Hirata, Y. (2000). Reentry into the cell cycle of contact-inhibited vascular endothelial cells by a phosphatase inhibitor. Possible involvement of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. J. Biol. Chem. 275, 3637–44.
Verbin, R.S. & Farber, E. (1967). Effect of cycloheximide on the cell cycle of the crypts of the small intestine of the rat. J. Cell Biol. 35, 649–58.
Vignon, X., Chesne, P., Le Bourhis, D., Flechon, J.E., Heyman, Y. & Renard, J.P. (1998). Developmental potential of bovine embryos reconstructed from enucleated matured oocytes fused with cultured somatic cells. C R Acad. Sci. III 321, 735–45.
Wakayama, T., Perry, A.C., Zuccotti, M., Johnson, K.R. & Yanagimachi, R. (1998). Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–74.
Wells, D.N., Laible, G., Tucker, F.C., Miller, A.L., Oliver, J.E., Xiang, T., Forsyth, J.T., Berg, M.C., Cockrem, K., L'Huillier, P.J., Tervit, H.R. & Oback, B. (2003). Coordination between donor cell type and cell cycle stage improves nuclear cloning efficiency in cattle. Theriogenology 59, 4559.
Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. & Campbell, K.H. (2007). Viable offspring derived from fetal and adult mammalian cells. Cloning Stem Cells 9, 37.



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