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Construction and Validation of Rabbit Oct4 Promoter-Driven RFP Expression Vector
Tang Lan1, Jiang Weihua2, Yin Mingru2, Zhou Xiaomei1, Li Heping1*, Chen Xuejin2, Li Shangang2*
1College of Wildlife Resources, Northeast Forestry University, Harbin 150040, China; 2Department of Laboratory Animal Sciences,Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
Abstract: The OCT4 transcription factor plays an important role in maintaining， regulating and controlling the pluripotency of embryonic stem cells/ES cells. The Oct4 gene promoter driving the expression of marker protein is significant for the pluripotency research of ES cells and the establishment of induced pluripotent stem cells/iPS cells. The green fluorescent protein (GFP) is always used as transfection marker in lentivirus transfection process， so the rabbit Oct4 gene promoter driving the expression of red fluorescent protein (RFP) is planned to be established， which will be useful to the study on rabbit ES cells and iPS cells preparation. The Oct4 promoter of rabbit (rOct4) was cloned through PCR and the expression vector， rOct4-RFP was constructed as the RFP was driven by rOct4. The rOct4-RFP vector was proved to be correct by the mouse ES cells transfection and the rabbit fibroblast cell line was transfected by rOct4-RFP to obtain rabbit rOct4-RFP fibroblasts. By enzyme digestion and sequencing verification， rOct4-RFP construction is not only proved to be successful， but also is able to express the red fluorescent protein in mouse ES cell line， E14. The red fluorescent protein can’t be expressed in differentiated rOct4-RFP transgenic E14 cells. The rOct4-RFP transgenic fibroblast cells are established through liposome mediated transgenosis， resistance screening and identification of PCR.
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