Generation of induced pluripotent stem cells from endangered cattle breeds makes a significant contribution to the establishment of embryonic stem cell line, conservation and utilization of genetic resources in endangered cattle. In our study, we are trying to construct recombinant proteins of transcription factors Oct-4, Nanog, Sox2 and Lin28 for reprogramming of endangered Luxi cattle fibroblast cells in induced pluripotent stem cells. To test the cell permeability and stability of the recombinant proteins, we designed and fused a pep-1 protein transduction domain to the C-terminus of enhanced green fluorescent protein, we treated Luxi cattle fibroblast cells with the purified proteins at various concentrations by adding them to the cell culture media for 2-48 h and assayed cell morphology and protein presence. We found that the purified EGFP-tagged recombinant proteins readily entered cells at a concentration of 0.12 mg/ml within 2 h and some of them could translocate into nucleus. In addition, we found that the transduced proteins appeared to be stable inside cells for up to 48 h. Then codons of Oct-4, Nanog, Sox2 and Lin28 were optimized for high level of expression in E. coli., they were synthesized using DNA oligo based, PCR gene assembling method, the reprogramming factors were designed with optimized transcription factors fused a pep-1 protein transduction domain to the C-terminus, for high level protein expression of the reprogramming factors, inducer concentration, and induction time were tested, the optimum inducer concentration for the expression of reprogramming factors Oct-4, Nanog, Sox2 and Lin28 were 0.01 mM, the optimum induction time were 10, 8, 2 and 12 h, respectively.
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 × 10(6) 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.
Chemokines were a major regulator of body's inflammatory and immune responses. In this study, the cDNA fragment of chemokine CXC ligand 10 (CXCL10) was cloned from the Ujumqin sheep ear marginal tissue cDNA expression library; the CXCL10 gene had 103 amino acids and a molecular weight of 11.47 kDa, and it shared a high homology among cattle, sheep, and goat, while a low homology compared with mouse. The CXCL10 protein had 4 conservative cysteine residues, located in 28, 30, 55, and 72 sites. The expression pattern and intracellular distribution of recombinant CXCL10 proteins in Ujumqin sheep fibroblast cells showed that there were green fluorescence signals both in cytoplasm and nucleolus after 24 h of transfection, the number of positive cells was increased with time, the peak level of fluorescence signal was reached after 48 h of transfection and the transfection efficiency was 33.3%; there was a significant decrease in fluorescence intensity after 72 h of transfection. Expression of recombinant CXCL10 gene in Escherichia coli had a time- and temperature-dependency on the amount of protein expression, and a small quantity of inducer was needed.
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