Skip to main content Accessibility help
×
Home

Fibroblast cell line establishment, cryopreservation and interspecies embryos reconstruction in red panda (Ailurus fulgens)

  • Yong Tao (a1) (a2), Jianming Liu (a1) (a2), Yunhai Zhang (a1), Meiling Zhang (a1), Junshun Fang (a1), Wei Han (a1), Zhizhong Zhang (a3), Ya Liu (a1), Jianping Ding (a1) and Xiaorong Zhang (a4)...

Summary

In evolution, the red panda (Ailurus fulgens) plays a pivotal role in the higher level phylogeny of arctoides carnivore mammals. The red panda inhabits certain Asian countries only and its numbers are decreasing. Therefore, the development of feasible ways to preserve this species is necessary. Genetic resource cryopreservation and somatic cell nuclear transfer (SCNT) have been used extensively to rescue this endangered species. The present study describes the establishment, for the first time, of a red panda ear fibroblast cell line, which was then cryopreserved, thawed and cultured. Through micromanipulation, interspecies embryos were reconstructed using the cryopreserved–thawed fibroblasts of the red panda as the donor and rabbit oocytes as recipients. A total of 194 enucleated rabbit oocytes were reconstructed with red panda ear fibroblasts; enucleated oocytes were activated without fusion as the control. The results show that the fibroblast cell line was established successfully by tissue culture and then cryopreserved in liquid nitrogen. Supplementation with 20% fetal bovine serum and 8% dimethyl sulphoxide in basic medium facilitated the cryopreservation. The interspecies embryos were successfully reconstructed. The cleavage, morulae and blastocyst rates after in vitro culture were 71, 47 and 23% (31/194), respectively. This study indicated that a somatic cell line could be established and cryopreserved from red panda and that rabbit cytoplast supports mitotic cleavage of the red panda karyoplasts and is capable of reprogramming the nucleus to achieve blastocysts.

Copyright

Corresponding author

All correspondence to: Zhang Xiaorong. College of Animal Science and Technology, Anhui Agricultural University, Changjiang West Road 130, Hefei, 230036, China. Tel: +8 6551 5782 488. Fax: +8 6551 5785 543. e-mail: zxr@ahau.edu.cn

References

Hide All
Borderie, V.M., Lopez, M., Lombet, A., Carvajal-Gonzalez, S., Cywiner, C. & Laroche, L. (1998). Cryopreservation and culture of human corneal keratocytes. Invest. Ophthalmol. Vis. Sci. 39, 1511–9.
Chen, D.Y., Sun, Q.Y., Liu, Y.L., Li, G.P., Lian, L., Wang, M.K., Han, M.Z., Song, X.F. & Sun, Q. (1999). The giant panda (Ailuropoda melanoleuca) somatic nucleus can dedifferentiate in rabbit ooplasm and support early development of the reconstructed egg. Sci. China (Ser C) 42, 346–53.
Choi, Y.H., Love, C.C., Chung, Y.G., Varner, D.D., Westhusin, M.E., Burghardt, R.C. & Hinrichs, K. (2002). Production of nuclear transfer horse embryos by piezo-driven injection of somatic cell nuclei and activation with stallion sperm cytosolic extract. Biol. Reprod. 67, 561–7.
Dominko, T., Mitalipova, M., Haley, B., Beyhan, Z., Memili, E., McKusick, B. & First, N.L. (1999). Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species. Biol. Reprod. 60, 1496–502.
Flynn, J.J., Nedbal, M.A., Dragoo, J.W. & Honeycutt, R.L. (2000). Whence the red panda? Mol Phylogenet. Evo. 17, 190–9.
Freshney, R.I. Disaggregation of the tissue and primary culture. (1994). In: Freshney, RI (ed.)., Culture of animal cells: a manual of basic technique. New York: Wiley-Liss & Sons., Inc. pp. 127–47.
Hayes, O., Rodriguez, L., Gonzalez, A., Falcon, V., Aguilar, A. & Castro, F.O. (2005). Effect of cryopreservation on fusion efficiency and in vitro development into blastocysts of bovine cell lines used in somatic cell cloning. Zygote 13, 277–82.
Ikumi, S., Sawai, K., Takeuchi, Y., Iwayama, H., Ishikawa, H., Ohsumi, S. & Fukui, Y. (2004). Interspecies somatic cell nuclear transfer for in vitro production of Antarctic minke whale (Balaenoptera bonaerensis) embryos. Cloning Stem Cells 6, 284–93.
Jiang, M.X., Yang, C.X., Zhang, L.S., Zheng, Y.L., Liu, S.Z., Sun, Q.Y. & Chen, D.Y. (2004). The effects of chemical enucleation combined with whole cell intracytoplasmic injection on panda–rabbit interspecies nuclear transfer. Zygote 12 (4), 315–20.
Kearney, J.N.Cryopreservation of cultured skin cells. (1991). Burns 1991., 17, 380–3.
Lanza, R.P., Cibelli, J.B., Diaz, F., Moraes, C.T., Farin, P.W., Farin, C.E., Hammer, C.J., West, M.D. & Damiani, P. (2000). Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer. Cloning 2, 7990.
Lee, B., Wirtu, G.G., Damiani, P., Pope, E., Dresser, B.L., Hwang, W. & Bavister, B.D. (2003). Blastocyst development after intergeneric nuclear transfer of mountain bongo antelope somatic cells into bovine oocytes. Cloning Stem Cells 5, 2533.
Li, J.S., Han, Z.M., Zhu, Z.Y., Wen, D.C., Liu, Z.H., Wang, M.K., Lian, L., Du, J., Wang, P.Y., Zhang, H.M. & Chen, D.Y. (2002). Interspecies nuclear transfer using nonquiescent somatic cell. J. Exp. Biol. 35, 62–5.
Li, M., Wei, F., Goossens, B., Feng, Z., Tamate, H.B., Bruford, M.W. & Funk, S.M. (2005). Mitochondrial phylogeography and subspecific variation in the red panda (Ailurus fulgens): implications for conservation. Mol. Phylogenet. Evol. 36, 7889.
Liu, S.Z., Zhou, Z.M., Chen, T., Zhang, Y.L., Wen, D.C., Kou, Z.H., Li, Z.D., Sun, Q.Y. & Chen, D.Y. (2004). Blastocysts produced by nuclear transfer between chicken blastodermal cells and rabbit oocytes. Mol. Reprod. Dev. 69, 296302.
Liu, Y., Zhang, X.R., Chen, D.Y., Zhang, Y.H., Zhang, Z.G., Jin, R.T., Wang, C.L., Zhang, M.L., Li, D.W., Li, B., Zhao, H. & Cheng, L.Z. (2003). In vitro developmental potential of cloned embryos derived from bovine somatic and rabbit oocyte. Agric. Sci. China 2, 1393–7.
Liu, Y., Zhang, X.R., Zhang, Y.H., Jin, R.T., Wang, C.L. & Wu, J.Y. (2004a). Storage and cryopreservation of bovine and goat fibroblast (in Chinese). Chin. J. Vet. Sci. 24, 197–8.
Liu, Y., Zhang, X.R., Chen, D.Y., Zhang, Y.H., Zhang, Z.G., Jin, R.T., Wang, C.L., Zhang, M.L., Li, D.W., Lin, B., Zhao, H. & Cheng, L.Z. (2004b). Study on development of cloned embryo using bovine somatic cell and rabbit oocyte in vitro. Scientia Agricultura Sinica 2004b; 37, 441–5.
Loi, P., Ptak, G., Barboni, B., Fulka, J., Cappai, P. & Clinton, M. (2001). Genetic rescue of an endangered mammal by cross species nuclear transfer using post-mortem somatic cells. Nature Biotech. 19, 962–4.
Lu, F., Shi, D; Wei, J., Yang, S. & Wei, Y. (2005). Development of embryos reconstructed by interspecies nuclear transfer of adult fibroblasts between buffalo (Bubalus bubalis) and cattle (Bos indicus). Theriogenology 64, 1309–19.
Murakami, M., Otoi, T., Wongsrikeao, P., Agung, B., Sambuu, R. & Suzuki, T. (2005). Development of interspecies cloned embryos in yak and dog. Cloning Stem Cells 7, 7781.
Peura, T.T. (2000). Serum starvation can cause excessive DNA damage in sheep fetal fibroblasts. Theriogenology 55, 285 (Abstract).
Peura, T.T., Hartwich, K.M., Hamilton, H.M. & Walker, S.K. (2003). No differences in sheep somatic cell nuclear transfer outcomes using serum-starved or actively growing donor granulosa cells. Reprod. Fertil. Dev. 15, 157–65.
Sansinena, M.J., Hylan, D., Hebert, K., Denniston, R.S. & Godke, R.A. (2005). Banteng (Bos javanikus) embryos and pregnancies produced by interspecies nuclear transfer. Theriogenology 63, 1081–91.
Slattery, J.P. & O'Brien, S.J. (1995). Molecular phylogeny of the red panda (Ailurus fulgens). J. Hered. 86, 413–22.
Tani, T., Kate, K. & Tsunoda, Y. (2000). Developmental potential of cumulus cell-derived culture frozen in a quiescent state nuclear transfer. Theriogenology 53, 1623–9.
Thongphakdee, A., Numchaisrika, P., Omsongkram, S., Chatdarong, K., Kamolnorranath, S., Dumnui, S. & Techakumphu, M. (2006). In vitro development of marbled cat embryos derived from interspecies somatic cell nuclear transfer. Reprod. Dom. Anim. 41, 219–26.
Tian, Y., Nie, W.H., Wang, J.H., Yang, Y.F. & Yang, F.T. (2002). Comparative chromosome painting shows the red panda (Ailurus fulgens) has a highly conserved karyotype. Acta Genetic Sinica 29, 124–7.
Wen, D.C., Yang, C.X., Cheng, Y., Li, J.S., Liu, Z.H., Sun, Q.Y., Zhang, J.X., Lei, L., Wu, Y.Q., Kou, Z.H. & Chen, D.Y. (2003). Comparison of developmental capacity for intra- and interspecies cloned cat (Felis catus) embryos. Mol. Reprod. Dev. 66, 3845.
Wen, D.C., Bi, C.M. & Chen, D.Y. (2004). Progress in interspecies cloning of mammals. Prog. Natur. Sci. 14, 18.
Wen, D.C., Bi, C.M., Xu, Y., Yang, C.X., Zhu, Z.Y., Sun, Q.Y. & Chen, D.Y. (2005). Hybrid embryos produced by transferring panda or cat somatic nuclei into rabbit MII oocytes can develop to blastocyst in vitro. J. Exp. Zool. 303, 689–97.
White, K.L., Bunch, T.D., Mitalipov, S. & Reed, W.A. (1999). Establishment of pregnancy after the transfer of nuclear transfer embryos produced from the fusion of argali (Ovis ammon) nuclei into domestic sheep (Ovis aries) enucleated oocytes. Cloning 1, 4754.
Zhang, X.R., Liu, Y., Zhang, Y.H., Chen, D.Y., Lian, L., Li, J.S., Jin, R.T., Zhang, Z.G., Zhang, M.L. & Wang, C.L. (2004). Effect of some factors on the fusion rate of bovine–rabbit interspecies reconstructed eggs. Chin. J. Agric. Biotech. 1, 135–8.
Zhang, Y.H., Pan, D.K., Sun, X.Z., Sun, G.J., Wang, X.B., Liu, X.H., Li, Y., Dai, Y.P. & Li, N. (2006). Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer. Sci. Chin. (Series C) 149, 111.
Zhang, Z.G., Zhang, X.R., Liu, Y., Jin, R.T., Zhang, M.L., Wang, C.L. & Zhao, H. (2005). Study of serial nuclear transfer on goat (Bore) rabbit interspecies cloned embryo. Scientia Agricultura Sinica 38, 601–5.
Zhong, Z.S., Zhang, G., Meng, X.Q., Zhang, Y.L., Chen, D.Y., Schatten, H. & Sun, Q.Y. (2005). Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos. Exp. Cell Res. 306, 3546.

Keywords

Fibroblast cell line establishment, cryopreservation and interspecies embryos reconstruction in red panda (Ailurus fulgens)

  • Yong Tao (a1) (a2), Jianming Liu (a1) (a2), Yunhai Zhang (a1), Meiling Zhang (a1), Junshun Fang (a1), Wei Han (a1), Zhizhong Zhang (a3), Ya Liu (a1), Jianping Ding (a1) and Xiaorong Zhang (a4)...

Metrics

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed