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| Construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice |
| WANG Jing1, HU Yan1, TAN Bi-qin1, WANG Jia-jia1, ZHAO Meng-ting1, WENG Qin-jie2, ZHU Di-feng2, WANG Hui-ying3 |
1. Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China;
2. Center for Drug Safty Evaluation Research, College of Pharmacetical Sciences, Zhejiang University, Hangzhou 310058, China;
3. Department of Allergy, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China |
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Abstract Objective:To construct the expression vector pLCK-CD69-IRES-EGFP that contains mouse cell surface activation protein CD69 and enhanced green fluorescent protein(EGFP),and to generate CD69 transgenic mice based on this vector. Methods:First, RNA was extracted from mouse lung tissue and cDNA was synthesized via reverse transcription. PCR primer was designed through the PubMed searching, then mouse CD69 DNA fragment was amplified with PCR. Second, this DNA fragment was subcloned to the pInsulater-LCK-IRES-EGFP plasmid and constructed the transgenic vector after the verification of nucleotide sequence. Third, the expression vector was then transfected into 293 T cells and its expression in 293 T cells was observed under fluorescence microscope. Last, microinjection was performed to transfer the expression vector pLCK-CD69-IRES-EGFP into fertilized eggs, which were implanted into pseudo-pregnant recipient mice. After birth the tail samples of the pups were obtained for the purpose of genotyping to determine the transgenic founders. Fluorescence microscope and flow cytometer were used to measure the expression of CD69 on cells. Results:The construction of the expression vector pLCK-CD69-IRES-EGFP was verified by enzyme digestion and DNA sequencing. The transfected 293 T cell showed expression of the protein under fluorescence microscope. Identification of PCR for the tail tissue of the pups confirmed the present of CD69 transgene and resting lymphocytes demonstrated the expression of CD69. Conclusion:The construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice have been successfully processed, which lays a foundation of the solid pattern studies in inflammatory diseases.
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Received: 11 March 2015
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小鼠pLCK-CD69-IRES-EGFP表达载体构建及CD69转基因小鼠的建立
目的:构建携小鼠细胞表面活化蛋白CD69和增强型绿色荧光蛋白(EGFP)在细胞内核糖体切入位点(IRES)的表达载体pLCK-CD69-IRES-EGFP,并基于此创建CD69转基因小鼠。方法:首先通过小鼠肺组织提取RNA,逆转录成为cDNA,经过PubMed搜索设计PCR引物,PCR法扩增mCD69片断,接着将该DNA片段经测序验证后接入pInsulater-LCK-IRES-EGFP质粒,构建pLCK-CD69-IRES-EGFP转基因载体;再将其转染到293T细胞中,通过荧光显微镜技术确定其在293T细胞中的表达状况;最后将载体显微注射入受精卵并移植入假孕母小鼠,出生小鼠取尾经PCR鉴定获得阳性首建鼠,并通过荧光显微镜和流式细胞术确定淋巴细胞上CD69的表达。结果:经酶切、DNA测序鉴定证实pLCK-CD69-IRES-EGFP表达载体构建成功;荧光倒置显微镜检查证实其在转染的293T细胞内的蛋白表达;小鼠取尾PCR鉴定明确CD69转基因小鼠创建成功;CD69转基因小鼠有外周血淋巴细胞的减少及静息状态下CD69的表达。结论:成功构建pLCK-CD69-IRES-EGFP表达载体及制备CD69转基因小鼠,为CD69在炎症性疾病中作用的整体模型研究奠定了基础。
关键词：
抗原,
核糖体,
绿色荧光蛋白质类/代谢,
质粒,
转染,
小鼠,
转基因
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