Abstract
BackgroundRenilla 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.ResultsDLD1 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.ConclusionsBased 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.
Highlights
Renilla Luciferase reporter gene GL4.82 and GL4.13 promoter are key player in transfection, but precise knowledge of its targets in colon cancer remains limited
Characterization of the techniques DLD1 cells were transfected with a SV40-RLuc carrying Renilla luciferase under the control of the SV-40 promoter, using two different techniques
To characterize the best technique among both techniques used for transfection (ExGen500 and Electroporation) in this study, substrate coelenterazine 50 μg/ml was added to each well and output was recorded, using in vivo Imaging System (IVIS) machine with in 10 second of the substrate addition
Summary
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. Since early 1990 new drug developments have moved from general cytotoxic agents to molecular target directed therapeutic, eventually, there was a need to identify tumor types and individual patient tumors that express the target and could benefit from therapies in clinical trials, in vivo models used in preclinical development should be oriented, disease-to-disease or target directed [7]. This is still less clear and point of debate that which is the best model to use with respect to predict for clinical trails? In Biomedical sciences use of animals as models to help understand and predict responses in humans, in toxicology and pharmacology in particular remains both the major tool for biomedical advances and a source of significant controversy [8]
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