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Transgene expression of enhanced green fluorescent protein in cloned rabbits generated from in vitro-transfected adult fibroblasts

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Abstract

Live rabbits have previously been generated through nuclear transfer using adult cells as nuclear donors. We demonstrated in this study that transfected adult rabbit fibroblasts are also capable of supporting full-term development. The fibroblasts were transfected with a pEGFP-C1 plasmid using lipofectamine™ 2000, and the transgenic cells were derived from conditioned medium. The transgenic fibroblasts were cultured until confluent and then serum-starved prior to be used as nuclear donors. After nuclear transfer and activation, 22% (12/55) of the transgenic cloned embryos developed to the blastocyst stage. A total of 114 embryos at the 4- to 8-cell stage were transferred to the oviducts of 8 pseudo-pregnant mothers; 5 of these animals became pregnant, and 3 of the 5 mother rabbits carried the pregnancy to term. Caesarean section was performed on the 3 pregnant mothers, yielding 4 kits, one of which has survived for more than 9 months. Green fluorescence could be detected in the toenails of the living cloned rabbit and the offspring from the living cloned rabbit under ultraviolet light. DNA analyses confirmed that all 4 cloned rabbits were genetically identical to the transgenic donor cells, and that they all carried the EGFP gene. The present study demonstrated that transgenic rabbits can be generated through nuclear transfer. These results may facilitate future developments in the genetic engineering of rabbits.

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Acknowledgments

We thank Prof. Eckhard Wolf and Dr. Valeri Zakhartchenko for critical reading of the manuscript and suggestions for improvement of the photos. The study was supported by grants from the National Natural Science Foundation of China (No: 30040003), the National Transgenic Foundation of China (2007AA10Z1A1), the China Postdoctoral Science Foundation (20060400541) and Shanghai Leading Academic Discipline Project (S30201).

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Authors and Affiliations

  1. National Center for Molecular Genetics and Breeding of Animal, Institute of Animal Sciences, Chinese Academy of Agriculture Sciences, 100094, Beijing, China

    Shangang Li, Chunguang Yin, Lingyang Xu, Tao Liu, Hongbin Li & Lixin Du

  2. Department of Laboratory Animal Sciences, School of Medicine, Shanghai Jiao Tong University, 200025, Shanghai, China

    Yi Guo, Jianjun Shi, Fengying Xing, Yao Li & Xuejin Chen

  3. College of Animal Medicine, Xinjiang Agricultural University, 830052, Wulumuqi, China

    Chuanshan Zhang

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  1. Shangang Li
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  2. Yi Guo
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  3. Jianjun Shi
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  9. Yao Li
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  10. Hongbin Li
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Correspondence to Lixin Du or Xuejin Chen.

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Li, S., Guo, Y., Shi, J. et al. Transgene expression of enhanced green fluorescent protein in cloned rabbits generated from in vitro-transfected adult fibroblasts. Transgenic Res 18, 227–235 (2009). https://doi.org/10.1007/s11248-008-9227-y

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  • DOI: https://doi.org/10.1007/s11248-008-9227-y

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