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In vitro developmental competence of pig nuclear transferred embryos: effects of GFP transfection, refrigeration, cell cycle synchronization and shapes of donor cells

Published online by Cambridge University Press:  01 August 2006

Yun-Hai Zhang ,
Deng-Ke Pan ,
Xiu-Zhu Sun ,
Guo-Jie Sun ,
Xiao-Hui Liu ,
Xiao-Bo Wang ,
Xing-Hua Tian ,
Yan Li ,
Yun-Ping Dai  and
Ning Li
Yun-Hai Zhang
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Deng-Ke Pan
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China. College of Animal Science and Technology, China Agricultural University, Beijing 100094, P. R. China.
Xiu-Zhu Sun
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Guo-Jie Sun
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Xiao-Hui Liu
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Xiao-Bo Wang
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Xing-Hua Tian
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China. College of Animal Science and Technology, China Agricultural University, Beijing 100094, P. R. China.
Yan Li
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Yun-Ping Dai
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
Ning Li*
Affiliation:
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, P. R. China.
*
All correspondence to: Ning Li, State Key Laboratory for Agrobiotechnology, China Agricultural University, No. 2 Yuan-ming-yuan west Road, Haidian, Beijing 100094, P. R. China. Tel: +86 10 62731146. Fax: +86 10 62733904. e-mail: ninglbau@public3.bta.net.cn
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Summary

The present study was designed to evaluate the feasibility of producing pig transgenic blastocysts expressing enhanced green fluorescent protein (GFP) and to examine the effects of shape and preparation methods of donor cells on in vitro developmental ability of pig nuclear transferred embryos (NTEs). In experiment 1, the effect of GFP transfection on development of pig NTEs was evaluated. The cleavage and blastocyst rates showed no significant difference between NTEs derived from transfected and non-transfected donors. In experiment 2, the effect of different nuclear donor preparation methods on in vitro development of NTEs was examined. The cleavage rate showed no statistically significant differences among three preparation methods. The blastocyst rates of donor cells treated once at −4 °C and those of freshly digested cells were similar to each other (26.3% vs 17.9%). The lowest blastocyst rates (5.88%) were observed when cells cryopreserved at −196 °C were used as donors. In experiment 3, the effect of different cell cycle synchronization methods on the in vitro development potential of pig NTEs was evaluated. The cleavage rate of NTEs derived from cycling cells was much better than that of NTEs derived from serum-starved cells (64.4% vs 50.5%, p < 0.05), but no significant difference was observed between the the blastocyst rates of the two groups. In experiment 4, the effect of different shapes of cultured fibroblast cells on the in vitro development of pig NTEs was examined. The fusion rate for couplets derived from rough cells was poorer than that observed in couplets derived from round smooth cells (47.8% vs 76.8%, p < 0.05). However, there were no significant differences observed in the cleavage rate and blastocyst rate. In conclusion, the present study indicated that (i) refrigerated pig GFP-transfected cells could be used as donors in nuclear transfer and these NTEs could be effectively developed to blastocyst stage; (ii) serum starvation of GFP-transfected cells is not required for preimplantation development of pig NTEs; and (iii) a rough surface of GFP-transfected donor cells affects fusion rate negatively but has no influence on the cleavage rate or blastocyst rate of pig NTEs.

Keywords

Cell refrigerationGFPPigPreimplantational developmentSomatic nuclear transfer
Type
Research Article
Information
Zygote , Volume 14 , Issue 3 , August 2006 , pp. 239 - 247
Copyright
Copyright © Cambridge University Press 2006

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References

Arat, S., Gibbons, J., Rzucidlo, S.J., Respess, D.S., Tumlin, M. & Stice, S.L. (2002). In vitro development of bovine nuclear transfer embryos from transgenic clonal lines of adult and fetal fibroblast cells of the same genotype. Biol. Reprod. 66, 1768–74.CrossRefGoogle ScholarPubMed
Baguisi, A., Behboodi, E., Melican, D.T., Pollock, J.S., Destrempes, M.M., Cammuso, C., Williams, J.L., Nims, S.D., Porter, C.A., Midura, P., Palacios, M.J., Ayres, S.L., Denniston, R.S., Hayes, M.L., Ziomek, C.A., Meade, H.M., Godke, R.A., Gavin, W.G., Overstrom, E.W. & Echelard, Y. (1999). Production of goats by somatic cell nuclear transfer. Nat. Biotechnol. 17, 456–61.CrossRefGoogle ScholarPubMed
Betthauser, J., Forsberg, E., Augenstein, M., Childs, L., Eilertsen, K., Enos, J., Forsythe, T., Golueke, P., Jurgella, G., Koppang, R., Lesmeister, T., Mallon, K., Mell, G., Misica, P., Pace, M., Pfister-Genskow, M., Strelchenko, N., Voelker, G., Watt, S., Thompson, S. & Bishop, M. (2000). Production of cloned pigs from. in vitro systems. Nat. Biotechnol. 18, 1055–9.Google Scholar
Bondioli, K., Ramsoondar, J., Williams, B., Costa, C. & Fodor, W. (2001). Cloned pigs generated from cultured skin fibroblasts derived from a H-transferase transgenic boar. Mol. Reprod. Dev. 60, 189–95.CrossRefGoogle ScholarPubMed
Boquest, A.C., Day, B.N. & Prather, R.S. (1999). Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells. Biol. Reprod. 60, 1013–19.CrossRefGoogle ScholarPubMed
Boquest, A.C., Grupen, C.G., Harrison, S.J., McIlfatrick, S.M., Ashman, R.J., d'Apice, A.J. & Nottle, M.B. (2002). Production of cloned pigs from cultured fetal fibroblast cells. Biol. Reprod. 66, 1283–7.CrossRefGoogle ScholarPubMed
Chesne, P., Adenot, P.G., Viglietta, C., Baratte, M., Boulanger, L. & Renard, J.P. (2002). Cloned rabbits produced by nuclear transfer from adult somatic cells. Nat. Biotechnol. 20, 366–9.CrossRefGoogle ScholarPubMed
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.CrossRefGoogle ScholarPubMed
Cibelli, J.B., Grant, K.A., Chapman, K.B., Cunniff, K., Worst, T., Green, H.L., Walker, S.J., Gutin, P.H., Vilner, L., Tabar, V., Dominko, T., Kane, J., Wettstein, P.J., Lanza, R.P., Studer, L., Vrana, K.E. & West, M.D. (2002). Parthenogenetic stem cells in nonhuman primates. Science 295, 819.CrossRefGoogle ScholarPubMed
Dai, Y., Vaught, T.D., Boone, J., Chen, S.H., Phelps, C.J., Ball, S., Monahan, J.A., Jobst, P.M., McCreath, K.J., Lamborn, A.E., Cowell-Lucero, J.L., Wells, K.D., Colman, A., Polejaeva, I.A. & Ayares, D.L. (2002). Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nat. Biotechnol. 20, 251–5.CrossRefGoogle ScholarPubMed
De Sousa, P.A., Dobrinsky, J.R., Zhu, J., Archibald, A.L., Ainslie, A., Bosma, W., Bowering, J., Bracken, J., Ferrier, P.M., Fletcher, J., Gasparrini, B., Harkness, L., Johnston, P., Ritchie, M., Ritchie, W.A., Travers, A., Albertini, D., Dinnyes, A., King, T.J. & Wilmut, I. (2002). Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy. Biol. Reprod. 66, 642–50.Google Scholar
Enright, B.P., Jeong, B.S., Yang, X. & Tian, X.C. (2003a). Epigenetic characteristics of bovine donor cells for nuclear transfer: levels of histone acetylation. Biol. Reprod. 69, 1525–30.CrossRefGoogle ScholarPubMed
Enright, B.P., Kubota, C., Yang, X. & Tian, X.C. (2003b). Epigenetic characteristics and development of embryos cloned from donor cells treated by trichostatin A or 5-aza-2'-deoxycytidine. Biol. Reprod. 69, 896–901.CrossRefGoogle ScholarPubMed
Evans, M.J. & Kaufman, M.H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–6.CrossRefGoogle ScholarPubMed
Guo, J.T., An, Z.X., Li, Y., Li, X.F., Li, Y.Q., Guo, Z.K. & Zhang, Y. (2002). Cloned goats (Capra hircus) from adult ear cells. Science in China (Series C) 45, 260–7.Google Scholar
Hyun, S., Lee, G., Kim, D., Kim, H., Lee, S., Nam, D., Jeong, Y., Kim, S., Yeom, S., Kang, S., Han, J., Lee, B. & Hwang, W. (2003). Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein. Biol. Reprod. 69, 1060–8.CrossRefGoogle ScholarPubMed
Koo, D.B., Kang, Y.K., Choi, Y.H., Park, J.S., Han, S.K., Park, I.Y., Kim, S.U., Lee, K.K., Son, D.S., Chang, W.K. & Han, Y.M. (2000). In vitro development of reconstructed porcine oocytes after somatic cell nuclear transfer. Biol. Reprod. 63, 986–92.CrossRefGoogle ScholarPubMed
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, 412–19.Google Scholar
Kühholzer, B., Hawley, R.J., Lai, L., Kolber-Simonds, D. & Prather, R.S. (2001). Clonal lines of transgenic fibroblast cells derived from the same fetus result in different development when used for nuclear transfer in pigs. Biol. Reprod. 64, 1695–8.CrossRefGoogle ScholarPubMed
Lai, L. & Prather, R.S. (2003). Creating genetically modified pigs by using nuclear transfer. Reprod. Biol. Endocrinol. 1, 82.CrossRefGoogle ScholarPubMed
Lai, L., Kolber-Simonds, D., Park, K.W., Cheong, H.T., Greenstein, J.L., Im, G.S., Samuel, M., Bonk, A., Rieke, A., Day, B.N., Murphy, C.N., Carter, D.B., Hawley, R.J. & Prather, R.S. (2002a). Production of alpha-1,3-galacto-syltransferase knockout pigs by nuclear transfer cloning. Science 295, 1089Tao92.Google Scholar
Lai, L., Park, K.W., Cheong, H.T., Kuhholzer, B., Samuel, M., Bonk, A., Im, G.S., Rieke, A., Day, B.N., Murphy, C.N., Carter, D.B. & Prather, R.S. (2002b). Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells. Mol. Reprod. Dev. 62, 300–6.CrossRefGoogle ScholarPubMed
Lee, J.W., Wu, S.C., Tian, X.C., Barber, M., Hoagland, T., Riesen, J., Lee, K.H., Tu, C.F., Cheng, W.T. & Yang, X. (2003). Production of cloned pigs by whole-cell intracytoplasmic microinjection. Biol. Reprod. 69, 995–1001.Google Scholar
Liu, J.L., Wang, M.K., Sun, Q.Y., Zhang, X.R., Jiang, L.K. & Chen, D.Y. (2001). Refrigeration of donor cells in preparation for bovine somatic nuclear transfer. Reproduction 122, 801–8.CrossRefGoogle ScholarPubMed
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.Google Scholar
Park, K.W., Cheong, H.T., Lai, L., Im, G.S., Kuhholzer, B., Bonk, A., Samuel, M., Rieke, A., Day, B.N., Murphy, C.N., Carter, D.B. & Prather, R.S. (2001). Production of nuclear transfer-derived swine that express the enhanced green fluorescent protein. Anim. Biotechnol. 12, 173–81.CrossRefGoogle ScholarPubMed
Polejaeva, I.A., Chen, S.H., Vaught, T.D., Page, R.L., Mullins, J., Ball, S., Dai, Y., Boone, J., Walker, S., Ayares, D.L., Colman, A. & Campbell, K.H. (2000). Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407, 86–90.CrossRefGoogle ScholarPubMed
Prather, R.S., Hawley, R.J., Carter, D.B., Lai, L. & Greenstein, J.L. (2003). Transgenic swine for biomedicine and agriculture. Theriogenology 59, 115–23.CrossRefGoogle ScholarPubMed
Ramsoondar, J.J., Machaty, Z., Costa, C., Williams, B.L., Fodor, W.L. & Bondioli, K.R. (2003). Production of alpha 1,3-galactosyltransferase-knockout cloned pigs expressing human alpha 1,2-fucosylosyltransferase. Biol. Reprod. 69, 437–45.Google Scholar
Tao, T., Boquest, A.C., Machaty, Z., Peterson, A.L., Day, B.N. & Prather, R. (2000). Development of pig embryos by nuclear transfer of cultured fibroblast cells. Cloning 1, 55–62.CrossRefGoogle Scholar
Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S. & Jones, J.M. (1998). Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–7.CrossRefGoogle ScholarPubMed
Uhm, S.J., Kim, N.H., Kim, T., Chung, H.M., Chung, K.H., Lee, H.T. & Chung, K.S. (2000). Expression of enhanced green fluorescent protein (EGFP) and neomycin resistant (Neo(R)) genes in porcine embryos following nuclear transfer with porcine fetal fibroblasts transfected by retrovirus vector. Mol. Reprod. Dev. 57, 331–7.Google Scholar
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.CrossRefGoogle ScholarPubMed
Walker, S.C., Shin, T., Zaunbrecher, G.M., Romano, J.E., Johnson, G.A., Bazer, F.W. & Piedrahita, J.A. (2002). A highly efficient method for porcine cloning by nuclear transfer using. in vitro-matured oocytes. Cloning Stem Cells 4, 105–12.CrossRefGoogle ScholarPubMed
Wang, Y.G., Zhou, X.G., Liu, J.L. et al. , (1999). Cloned goats (Capra hircus) from foetal fibroblast cell lines. Chinese Sci. Bull. 45, 34–8.CrossRefGoogle Scholar
Watanabe, S., Iwamoto, M., Suzuki, S., Fuchimoto, D., Honma, D., Nagai, T., Hashimoto, M., Yazaki, S., Sato, M. & Onishi, A. (2005). A novel method for the production of transgenic cloned pigs: electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin. Biol. Reprod. 72, 309–15.CrossRefGoogle ScholarPubMed
Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J. & Campbell, K.H. (1997). Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–13.Google Scholar
Yin, X.J., Tani, T., Yonemura, I., Kawakami, M., Miyamoto, K., Hasegawa, R., Kato, Y. & Tsunoda, Y. (2002). Production of cloned pigs from adult somatic cells by chemically assisted removal of maternal chromosomes. Biol. Reprod. 67, 442–6.CrossRefGoogle ScholarPubMed
Zhou, Q., Jouneau, A., Brochard, V., Adenot, P. & Renard, J.P. (2001). Developmental potential of mouse embryos reconstructed from metaphase embryonic stem cell nuclei. Biol. Reprod. 65, 412–19.CrossRefGoogle ScholarPubMed