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Derivation and characterization of goat fetal fibroblast cells induced with human telomerase reverse transcriptase

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Abstract

Fetal fibroblast cells (FFCs) are often used as donor cells for somatic cell nuclear transfer (SCNT) because they are easy to culture and suitable for genetic manipulation. However, through genetic modification process, which required FFCs to be cultured in vitro for several passages, cells tended to age very rapidly and became inappropriate for SCNT. Human telomerase reverse transcriptase (hTERT) possessed the activity of human telomerase and maintains telomere in dividing cells; therefore, hTERT can be transfected into somatic cells to extend their lifespan. In this study, we transfected a Xinong Saanen Dairy Goat FFC line with hTERT. Then, we tested several characteristics of transfected cells, including growth curve, expression and activity of hTERT, tumorigenicity, and expression of oct4 and nanog. The result showed that hTERT could significantly extend the lifespan of transfected cells in vitro. hTERT mRNA was expressed in hTERT-transfected cells. Moreover, hTERT-transfected cells presented enhanced telomerase activity and longer telomere than untransfected cells at the same passage. On the other hand, hTERT-transfected cells can maintain normal karyotype even after several times of subculture in vitro. After inoculation of hTERT-transfected cells in nude mouse, none of them developed tumors on the vaccination site. Interestingly, transfection of hTERT can improve expression of nanog and oct4 in Xinong Saanen Dairy Goat FFCs, especially in low generation after transfection, but with increasing subculture, this effect gradually weakened.

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Acknowledgments

We thank Professor Yaping Jin for providing the plasmid and Dr. Yajun Liu and Xiaojun Qu for their technical help. This study was supported by funds from the National Science and Technology Major Project of China (no. 2009ZX08008-010B) and State Key Laboratory of Veterinary Etiological Biology (SKLVE2009KFKT001).

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Authors and Affiliations

  1. College of Animal Veterinary Medicine, Northwest A&F University, Yangling, Xianyang, Shaanxi, 712100, China

    Ying Xie, Xiaoe Zhao, Hongxiang Jia & Baohua Ma

  2. Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Yangling, Xianyang, Shaanxi, 712100, China

    Ying Xie, Xiaoe Zhao, Hongxiang Jia & Baohua Ma

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  1. Ying Xie
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  2. Xiaoe Zhao
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  3. Hongxiang Jia
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  4. Baohua Ma
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Correspondence to Baohua Ma.

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Editor: T. Okamoto

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Fig. 1

Untransfected fibroblast at passage 30. Cells begin to deform, grow slowly, and exhibit senescence characteristics (JPEG 360 kb)

Fig. 2

Karyotype and tumorigenicity of hTERT fibroblast. a Karyotype was tested at 50th passage; b1 nude mouse 6 wk after inoculation of hTERT fibroblast at passage10; b2 nude mouse 6 wk after inoculation of hTERT fibroblast at passage30; b3 nude mouse 6 wk after inoculation of hTERT fibroblast at passage50; b4 nude mouse 6 wk after inoculation of Hela cells (JPEG 227 kb)

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Xie, Y., Zhao, X., Jia, H. et al. Derivation and characterization of goat fetal fibroblast cells induced with human telomerase reverse transcriptase. In Vitro Cell.Dev.Biol.-Animal 49, 8–14 (2013). https://doi.org/10.1007/s11626-012-9554-4

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  • DOI: https://doi.org/10.1007/s11626-012-9554-4

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