Siddique Muhammad Rashid, Shynder Steve, Ashraf Muhammad Aqeel, Yusoff Ismail, Wajid Abdul
Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia.
Chem Cent J. 2012 Jul 18;6(1):69. doi: 10.1186/1752-153X-6-69.
Renilla Luciferase reporter gene (rLuc) GL4.82 and GL4.13 promoter are key player in transfection, but precise knowledge of its targets in colon cancer remains limited. The aim of this study was to characterize the best transfection technique to produce a stable transfected colon DLD1 (colorectal adenocarcinoma cell line), therefore imaging based approaches were employed.
DLD1 cells were transfected with a Plasmid (SV40-RLuc) carrying Renilla luciferase under the control of the SV-40 promoter, by using two different transfection techniques. Cells expressing the required DNA were isolated after antibiotic (Puramycin) selection. Clones of DLD-1/SV40-RLuc were produced using two different techniques (96 well plates and Petri dish) and their florescence intensity was recorded using IVIS machine (Calliper Life Sciences, Hopkinton, USA). Both techniques were characterized with the help of serial dilution technique. Results from this study substantiated that electroporation is the best. As expected, clones varied in their specific luciferase activity along with the dilutions. With the increase in cell concentration increase in intensity of florescence was recorded.
Based on the results we are confident that this transfected cell line DLD-1/SV40-RLuc (colorectal adenocarcinoma cell line) is the best for further Orthotopic Xenotransplantation Studies and in-vivo experiments as well. Investigation shows that DLD1/SV-rLuc cells have gained little bit resistance against both drugs therefore further study is suggested to know the reasons.
海肾荧光素酶报告基因（rLuc）GL4.82和GL4.13启动子在转染中起关键作用，但对其在结肠癌中的靶点的精确了解仍然有限。本研究的目的是确定产生稳定转染的结肠癌细胞系DLD1（结直肠腺癌细胞系）的最佳转染技术，因此采用了基于成像的方法。
通过两种不同的转染技术，用携带在SV-40启动子控制下的海肾荧光素酶的质粒（SV40-RLuc）转染DLD1细胞。在抗生素（嘌呤霉素）选择后分离出表达所需DNA的细胞。使用两种不同技术（96孔板和培养皿）产生DLD-1/SV40-RLuc克隆，并使用IVIS成像系统（美国霍普金顿的 Caliper Life Sciences公司）记录其荧光强度。两种技术都借助系列稀释技术进行了表征。本研究结果证实电穿孔是最佳方法。正如预期的那样，克隆的特异性荧光素酶活性随稀释度而变化。随着细胞浓度的增加，荧光强度也增加。
基于这些结果，我们确信这种转染的细胞系DLD-1/SV40-RLuc（结直肠腺癌细胞系）最适合进一步的原位异种移植研究和体内实验。研究表明，DLD1/SV-rLuc细胞对两种药物都有一定的抗性，因此建议进一步研究以了解原因。
