Establishment of life-span extended bovine fibroblast cells carrying the characterization of primary cells

Seungkwon You, Minhee Heo, Jai Hee Moon, Sung Chan Kim, Sungwook Kwak, Du Hak Yoon, Dongil Jin, Ki Chang Hong, Douglas N. Foster, Yun Jaie Choi, Hyunggee Kim

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9 Citations (Scopus)

Abstract

Although primary bovine embryonic fibroblast (BEF) cells have previously been used as nucleus-donors for nuclear transfer (NT), it has now been proposed to use BEF cells to generate cloned cows that were genetically modified by transgenic or a knock-out system. A major limitation to gene targeting somatic cells, however, is the overall life-span of the cell. In this study, we first examined in vitro life-span of primary BEF cells. Primary BEF cells were found to be replicative senescent at passage 10th-12th, similar to primary murine embryonic fibroblast cells. To overcome this short in vitro life-span, we have optimized culture conditions to extend the life-span and determined growth characteristics of BEF cell lines. Two life-span extended BEF cell lines (designated CGFR -BO-1 and CGFR-BO-2) were shown to grow much faster than their parental primary counterparts. Both cell lines did not display any potential for abnormal growth such as foci formations in either soft-agar or confluent culture condition. In cloning experiments using these cell lines as a nuclear donor, the reconstructed karyoblasts underwent apoptosis, reprogramming and development in the blastocyst stage, at a similar frequency to those observed with parental as well as adult primary fibroblasts. Furthermore, these cell lines targeted with green fluorescence protein (GFP) were successfully transduced, selected and reprogrammed by NT to develop into a blastocyst stage with GFP expression. Our results suggested methods to extend life-span of donor cells with tremendous implications for the genetic engineering of bovine fibroblast cells.

Original languageEnglish
Pages (from-to)261-268
Number of pages8
JournalMolecules and Cells
Volume18
Issue number2
Publication statusPublished - 2004 Oct 31

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Keywords

  • Blastocyst
  • Bovine embryonic fibroblast
  • Cloning
  • Green fluorescent preotein
  • Life-span extended cells

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

You, S., Heo, M., Moon, J. H., Kim, S. C., Kwak, S., Yoon, D. H., Jin, D., Hong, K. C., Foster, D. N., Choi, Y. J., & Kim, H. (2004). Establishment of life-span extended bovine fibroblast cells carrying the characterization of primary cells. Molecules and Cells, 18(2), 261-268.