Part:BBa_K4419026
pCAG-loxP_NLS_NCre_IRES_GFP_loxP_mCherry (Mosquitoes)
Both C- and N-terminal region Cre enzymes co-operate to remove the sandwitched gene region between first and second lox P sequences, and then the red fluorescent protein (mCherry) is produced, although green fluorescent protein (EGFP) is produced before this removement.
Fig. 1.Confirmation of our construction by electrophoresis. Lane 1: λ/HindIII ; Lane 2: Backbone ; Lene 3: pCAG-loxP_NLS_NCre_IRES_GFP_loxP_mCherry (Mosquitoes) (~3.6K bp); Lene4:Product (~6.5K bp);
Transfection into cells
Two methods of transfection of cells were tried. Lipofection and electroporation. For electroporation, we used a one-cut restriction endonuclease on the plasmid and linearized the plasmid before transformation in order to achieve stable transformation. Due to a combination of experimental progress, we were able to try lipofection and electroporation for Vero cells and lipofection for C6/36 cells. These transformations should allow the cells to constantly express EGFP and N-Cre, and intracellular expression of C-Cre due to SRIP infection, etc., should lead to intracellular recombination and mCherry expression. In fact, we confirmed EGFP in Vero and C6/36 cells, and in Vero cells, EGFP was confirmed by both lipofection and electroporation.
Fig. 2.(A) C6/36 cells negative control fluorescent image, (B) C6/36 cells negative control bright-field image, (C) Lipofected C6/36 cells fluorescent image, (D) Lipofected C6/36 cells bright-field image ;
Sequence and Features| None |