소개글
KAIST cell biology experiment class 에서 작성한 세포생물학 실습, 실험 보고서입니다.
참고하시어 실험에 도움이 되시길 바랍니다.
논문형식으로 "영어로" 작성되었으며 이론과 결과를 매우 자세하게 작성하였습니다.
대장균과 벡터를 사용한 단백질의 발현에 관한 실험내용이니 세포생물학의 근본이 되는 주요 실험들을 모두 자세히 다루었습니다.
In this study, we constructed recombinant plasmid p53-EGFP with homosapiens tumor protein p53 and vector pEGFP-C1/N1. The purpose is confirming the efficiency of p53-EGFP expression in the mammalian cell, HEK 293T and HeLa cell.
목차
1. ABSTRACT
2. INTRODUCTION
3. RESULTS
4. DISCUSSION
5. MATERIALS AND METHODS
6. REFERENCE
본문내용
ABSTRACT
In this study, we constructed recombinant plasmid p53-EGFP with homosapiens tumor protein p53 and vector pEGFP-C1/N1. The purpose is confirming the efficiency of p53-EGFP expression in the mammalian cell, HEK 293T and HeLa cell. The amplified insert gene p53 is earn by PCR and with restriction enzyme HindⅢ and BamHⅠ, ligate vector pEGFP-C1/N1 and p53 gene by T4 ligase. The ligated plasmid seq is confirmed by gel electrophoresis and gene sequencing. We tried transformation the recombinant plasmid to E.coli to subcloning. Then, transfect with the plasmid to mammalian cell, HEK 293T. After the incubation, to confirm protein expression from plasmid, we did fluorescence microscope observation, western blot, immunocytochemistry, immunoprecipitation and FACS. We could see green florescence signal by fluorescence microscope observation, and by immunocytochemistry, we checked the presence of p53 and GFP with anti-rabbit 488 for GFP and anti-mouse 594 for p53. Also, by immunoprecipitation experiment, the p53-EGFP has expressed in fusion. Finally, with FACS, we tried to confirm expression efficiency but we couldn’t.
참고 자료
https://www.addgene.org/vectordatabase/2491/
Ying-Qiang Zhong, Wei J, Fu YR, Shao J, Liang YW, Lin YH, Liu J, Zhu ZH. Toxicity of cationic liposome Lipofectamine 2000 in human pancreatic cancer Capan-2 cells. n.p.: Journal of Southern Medical University, 1981-1984.
Sakari Hietanen, Sonia Lain, Eberhard Krausz, Christine Blattner, and David P. Lane. Activation of p53 in cervical carcinoma cells by small molecules. n.p.: , Harvard Medical School, Boston, MA, April 13, 2000.
Matlashewski et al. (1986). Eur. J. Biochem., 154, 665-672
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