Generating 4T1 stable cell line that expresses EGFP and enhanced Firefly Luciferase for the cancer research study.
Generating 4T1 stable cell line that expresses EGFP and enhanced Firefly Luciferase for the cancer research study.
Kaveh Nasrollahi,1Farangis Ataei,2,*Saman Hosseinkhani,3
1. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran 2. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran 3. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
Introduction: most developments in cancer research have happened with the help of animal models. animal models give us hints that can reach us to a cure for cancer. despite all of those advantages animal models have limitations that can be misleading. First of all, the immune cells work differently in animal models such as mice than in the human so isolation and characteristic of immune cells that work against tumor is an important part. Secondly, a big problem in the use of animal models is how the tumor develops and spreads in the body. One solution to this challenge is Bioluminescence imaging which is high-throughput, cheap, and scalable.
to answer those problems We have generated a 4T1 stable cell line that expresses enhanced firefly luciferase (fLuc+) and EGFP.
Methods: We have digested plex307-EGFP by BamHI that breaks upstream of the EGFP and then fLuc+-T2A has ligated into it. We have used plex307-fLuc+-T2A-EGFP, pspax, and pCAG-VSVG plasmids to produce lentiviruses that express EGFP and fLuc+. We transfected the 4T1 cell line with viruses and then do a selection by puromycine for two-week. finally, the single cell has been isolated from the pool by the limiting dilutions method.
Results: The fluc+ spectrum of the cell line confirms that the maximum emission of enhanced luciferase is 584nm. Also, EGPF expression has been confirmed by fluorescent microscopy.
Conclusion: The main purpose of present study is investigation of tumor developing in animal model
Keywords: 4t1, animal model, firefly luciferase, cancer, singel cell,