Designed by: Leonardo ken Okumura, Ryoko Enomoto   Group: iGEM22_TokyoTech   (2022-10-11)

pCAG-loxP_NLS_NCre_IRES_GFP_loxP_mCherry (Human)
In this study, we constructed the gene flow of LoxP_NLS_N-Cre_IRES_EGFP_LoxP_mCherry, which is the basis of SRIP detecting cell, by inserting two types of synthetic DNA into pIRES2-EGFP. Finally, LoxP_NLS_N-Cre_IRES_EGFP_LoxP_mCherry was inserted into pCAGGS as an insert, and this sequence was placed under the CAG promoter.
By doing so.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.Here we show N-Cre and mCherry codon-optimized for Human for transfection into Vero cells.

For confirmation, we compared the electrophoresis of insert (LoxP_NLS_N-Cre_IRES_EGFP_LoxP_mCherry) and backbone plasmid (pCAGGS) treated with two restriction enzymes EcoR I and Bgl II, and the product amplified in E. coli by ligation of these two enzymes.

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 (Human) (~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) Vero cells negative control fluorescent image, (B) Vero cells negative control bright-field image, (C) Lipofected vero cells fluorescent image, (D) Lipofected vero cells bright-field image, (E) Electroporated vero cells fluorescent image, (F) Electroporated vero cells bright-field image

In TokyoTech 2023, this composite part was used to transfect Vero cells with PEI (: Polyethylenimine). In the 2022 experiment, this parts were incorporated into linearized vectors and introduced into Vero cells by Electroporation and Lipofection. In the 2023 experiment, these parts were incorporated into a cyclized vector and introduced into Vero cells by PEI. This experiment demonstrated a new method of introducing this composite part into Vero cells.

Fig. 3.(A) Vero cells negative control fluorescent image, (B) Vero cells negative control bright-field image, (C) Transfected by PEI (: Polyethylenimine) vero cells fluorescent image, (D) Transfected by PEI (: Polyethylenimine) vero cells bright-field image; the scale bar is 50 µm.


Sequence and Features BBa_K4419025 SequenceAndFeatures