Study on characteristics of in vitro culture and intracellular transduction of exogenous proteins in fibroblast cell line of Liaoning cashmere goat
- 238 Downloads
Establishment of fibroblast cell lines of endangered goat breeds and research on the gene or protein functions based on the cells made a significant contribution to the conservation and utilization of genetic resources. In this study, a fibroblast cell line of Liaoning cashmere goat, frozen in 174 cryovials with 5 × 106 cells each, was successfully established from 60 goats ear marginal tissues using explant culture and cryopreservation techniques. Biological analysis of in vitro cultured cell line showed that, the cells were morphologically consistent with fibroblasts; the average viability of the cells was 94.9 % before freezing and 90.1 % after thawing; the growth process of cells was consisted of a lag phase, a logarithmic phase and a plateau phase; cell population doubling time was 65.5 h; more than 90 % of cells were diploid prior to the 6th generation; Neither microbial contamination nor cross-contamination was detected. To determine cell permeability, intracellular path and stability of exogenous proteins during the transduction, a TAT protein transduction domain was fused to the C-terminus of enhanced green fluorescent protein, the established fibroblast cell line was treated with the purified exogenous proteins at various concentrations by adding them to the cell culture media for 1–24 h and assayed cell morphology and protein presence, it was found that the purified exogenous proteins readily entered cells at a concentration of 0.1 mg/ml within 1.5 h and some of them could translocate into nucleus, moreover, the exogenous proteins appeared to be stable inside cells for up to 24 h.
KeywordsLiaoning cashmere goat Fibroblast cell line Exogenous protein TAT peptide Transduction
- 4.Gu Y, Li H, Miki J, Kim KH, Furusato B, Sesterhenn IA, Chu WS, McLeod DG, Srivastava S, Ewing CM, Isaacs WB, Rhim JS (2006) Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines. Exp Cell Res 312(6):831–843PubMedCrossRefGoogle Scholar
- 6.Kawarai S, Hashizaki K, Kitao S, Nagano S, Madarame H, Neo S, Ishikawa T, Furuichi M, Hisasue M, Tsuchiya R, Tsujimoto H, Yamada T (2006) Establishment and characterization of primary canine hepatocellular carcinoma cell lines producing alpha-fetoprotein. Vet Immunol Immunopathol 113(1–2):30–36PubMedCrossRefGoogle Scholar
- 7.Guan WJ, Ma YH, Zhou XY, Liu GL, Liu XD (2005) The establishment of fibroblast cell line and its biological characteristic research in Taihang black goat. Rev China Agric Sci Technol. 7:25–33Google Scholar
- 12.Ansari HA, Bosma AA, Broad TE, Bunch TD, Long SE, Maher DW, Pearce PD, Popescu CP (1999) Standard G-, Q-, and R-banded ideograms of the domestic sheep (Ovis aries): homology with cattle (Bos taurus). Report of the committee for the standardization of the sheep karyotype. Cytogenet Cell Genet 87(1–2):134–142PubMedCrossRefGoogle Scholar