摘要
[Objective] To clone ovarian-specific promoter-1 （OSP-1） fragment in rats and detect the tissue-specific expression of OSP-1 at cell level. [Method] According to the known OSP-1 sequence in rats, the specific pdmers were designed. The OSP-1 fragment was amplified by PCR and compared with published OSP-1 sequence. After eukaryotic expression vector pOSP-1-EGFP-N1 was constructed by cloning OSP-1 promoter into the pEGFP-N1 vector without CMV, the recombinant plasmid was transfected into buffalo granulocytes, mammary epithelial cells and fetal fibroblast cells under mediation of liposome LipofectamineTM 2000. The green fluxorescent protein （GFP） expression was observed with the fluorescence microscope after transfection for 12, 24 and 48 h. [Result] The amplified OSP-1 fragment was 480 bp, and the homology to published rat OSP-1 sequence was 96%. The sequence analysis showed that this fragment contained a core promoter cis-element similar with TATA box and ChAT box, and multiple C/EBP beta transcription factor binding sites. The EGFP-expressing positive granulccytes were firstly observed at 12 h after transfection and they were increased. All the EGFP-expressing and non-expressing cells from granulocytes were large and round. After transfection for 48 h, the EGFP-expressing positive granulocytes were decreased. The EGFP was not expressed in buffalo mammary epithelial cell and fetal fibroblast cell. [ Conclusion] EGFP exDression controlled bv OSP-1 promoter can be found in buffao granulocvtes.
[Objective] To clone ovarian-specific promoter-1 （OSP-1） fragment in rats and detect the tissue-specific expression of OSP-1 at cell level. [Method] According to the known OSP-1 sequence in rats, the specific pdmers were designed. The OSP-1 fragment was amplified by PCR and compared with published OSP-1 sequence. After eukaryotic expression vector pOSP-1-EGFP-N1 was constructed by cloning OSP-1 promoter into the pEGFP-N1 vector without CMV, the recombinant plasmid was transfected into buffalo granulocytes, mammary epithelial cells and fetal fibroblast cells under mediation of liposome LipofectamineTM 2000. The green fluxorescent protein （GFP） expression was observed with the fluorescence microscope after transfection for 12, 24 and 48 h. [Result] The amplified OSP-1 fragment was 480 bp, and the homology to published rat OSP-1 sequence was 96%. The sequence analysis showed that this fragment contained a core promoter cis-element similar with TATA box and ChAT box, and multiple C/EBP beta transcription factor binding sites. The EGFP-expressing positive granulccytes were firstly observed at 12 h after transfection and they were increased. All the EGFP-expressing and non-expressing cells from granulocytes were large and round. After transfection for 48 h, the EGFP-expressing positive granulocytes were decreased. The EGFP was not expressed in buffalo mammary epithelial cell and fetal fibroblast cell. [ Conclusion] EGFP exDression controlled bv OSP-1 promoter can be found in buffao granulocvtes.
基金
supported by the Major Projects of Genetically Modified Organism (GMO) New Varieties Breeding of Ministry of Agriculture ( 2008ZX08007-003)
