Establishment of life-span extended bovine fibroblast cells carrying the characterization of primary cells.
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.
You
Seungkwon
S
Laboratory of Cell Growth and Function Regulation, Korea University, Seoul 136-701, Korea. bioseung@korea.ac.kr
Heo
Minhee
M
Moon
Jai-Hee
JH
Kim
Sung-Chan
SC
Kwak
Sungwook
S
Yoon
Du-Hak
DH
Jin
Dongil
D
Hong
Ki-Chang
KC
Foster
Douglas N
DN
Choi
Yun-Jaie
YJ
Kim
Hyunggee
H
D000818:Animals; D017209:Apoptosis; D001757:Blastomeres / Q000166:cytology; D002417:Cattle; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D016922:Cellular Senescence; D019976:Cloning, Organism / Q000379:methods; D005347:Fibroblasts / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D053652:Nuclear Transfer Techniques; D014161:Transduction, Genetic
D049452:Green Fluorescent Proteins