From 2013.igem.org

Latest revision as of 02:41, 29 October 2013

Overview of Cre Recombinase

Taken from from the P1 Bacteriophage, Cre recombinase is widely used as a site specific DNA recombinase. The protein functions by binding to LoxP as a dimer, then two bound dimers coming together as a tetramer, creating a Holiday Junction, and finally breaking, recombining, and re-ligating the DNA back together (Sauer, 1998).

One useful property of Cre recombinase is its ability to recombine DNA at very low concentrations of active protein. Since the ultimate goal is to send Cre recombinase through exosomes, the amount of Cre recombinase entering the sender cell will likely be small, but as mentioned earlier, that shouldn't be a problem, as it doesn't take very many Cre molecules to trigger a recombination event.

Sauer, B & Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci. (1998)

Characterization

Before we can consider fusing Cre recombinase to Acyl-TyA, we must first determine that we can get unmodified Cre recombinase functioning in single cells, and second, determine that the Acyl-TyA-Cre fusion protein functions as well. The diagram below demonstrates how Cre recombinase activity will be assayed. When the reporter construct is transfected into cells, it produces red fluorescence, but when Cre recombinase is present, the LoxP sites flanking the RFP gene causes it to be spliced out by Cre and the reporter shifts from producing red fluorescence to producing green fluorescence from the GFP gene.

In order to test the function of Cre, we transfected the following circuit into HEK293 cells:

Positive Well:
The following well was seeded with 200,000 cells and the following DNA amounts: 0ng CMV_Cre, 500ng pCMV_(LoxP)RFP(LoxP)GFP (reporter) and 100ng hEF1a_tagBFP. The following images were gathered 72 hours post transfection.

Control Well:
The following well was seeded with 200,000 cells and the following DNA amounts: 0ng CMV_Cre, 500ng pCMV_(LoxP)RFP(LoxP)GFP (reporter) and 100ng hEF1a_tagBFP. The following images were gathered 72 hours post transfection.

As you can see from the images above, the control well demonstrated evidence of recombination events without the presence of Cre recombinase. New aliquots of the reporter construct were made to rule out the possibility of contamination, but results were similar. We've determined that the problem is either an issue with our reporter construct or an incredibly unlikely stable cell line integration of Cre, as we were transfecting in hoods which are used for lentiviral cell transduction. Despite this, we did get an interesting video of cells changing color on the confocal microscope (The are, unfortunately, changing color in the wrong direction).

Exosome Isolation and Application

This experiment will entail transfecting Jurkat T cells with hEF1a_Acyl-TyA-Cre, isolating exosomes from the media they were grown in, and treating HEK 293 receiver cells (transfected with the reporter construct) with the isolated exosomes. We expect to see the Acyl-TyA-Cre enter the reciever cells and recombine the reporter plasmid, causing the receivers to change from red to green.

Cell-Cell Coculturing

This experiment will involve the same HEK 293 receiver cells, but we will instead add different amounts of Jurkat T cells transfected with hEF1a_Acyl-TyA-Cre together with the receiver cells. We expect the Jurkats to produce exosomes containing Acyl-TyA-Cre which can then enter the receiver cells and trigger the reporter construct.