Is CAG a promoter, and the black tri-angle represents loxP site of site-specific recombination system Cre-loxP?

Yuan-Yeu Yau: yes, CAG is the promoter and the triangles are loxP sites, according to what described by the Jackson Lab (No. 017492)

Mai Dan Nguyen,

To understand those constructs in your question, you need to know the Cre-loxP site-specific recombination system. See attached figure. The Cre is the enzyme, and it can catalyze two identical loxP sites for recombination, also called "site-specific recombination".

Depending on the orientation of the two loxP sites, the gene (or a DNA fragment) placed in-between these two identical sites will be either 'reversed (flip)' or 'deleted' when Cre is expressed. If the gene/(dna fragment) is placed in two loxP sites with the same orientation, the gene/(dna fragment) will be delected upon the expression of Cre gene. If the two loxP sites are placed in opposite position, the gene/(dna fragment) in-between will 'flip' around until the Cre activity is abolished.

Go through this info, and think about how your constructs are formed after Cre is expressed. I will get back to you later.

There are more infromation about Cre-loxP system from this site:

https://www.addgene.org/cre-lox/

Ok, I attach a PPT for you. I assigned a number to each 'loxP' site (black triangle). There are 5 loxP sites on contruct 'X'.

1. For construct 'A': upon Cre expression, if the site-specific recombination occurs at loxP sites '1' and '2', the DNA fragment within these two sites will be deleted (because the loxP sites are placed at the same orientation) and lead to the generation of construct 'A'. After the removal of the fragment, promoter CAG will fuse to hrGFPII, and target to nucleus (so the figure show 'green color' in the nucleus.

2. For construct 'B': similarly, in this case, Cre protein works on the recombination between loxP '1" and loxP '4", where DNA fragment between these two sites will be deleted. After the removal of the DNA fragment, promoter CAG will fuse to RFP and target to somewhere in the cytoplasm of a cell (so the figure show 'red' color in the cytoplasma).

3. Could you figure out constructs 'C' and 'D' and let me know? (hint: here it involves 'DNA fragment inversion (or flip)' within two loxP sites)

Dear Yuan-Yeu Yau,

Thank you very much for your kind help and very clear explanation. For your question, construct "C" would be the inversion between loxP site 1 and 4, leading to the expression of cytoplasmic YFP. Similarly, cell membrane CFP would result from inversion of the whole locus, between loxP site 1 and 5, giving rise to construct D. But I also wonder if there are two recombination events occur at the same time, for example: Deletion of loxP site 1-2, then inversion of loxP site 2-3 to yield yellow color. And if this happens in real, there should be more than 4 possible constructs (but there are only 4 color outcomes), shouldn't there?

Yes, your answer is correct for how constructs 'C' and 'D' are derived from construct 'X'. And, you are right. Theoriotically, there can be more than 4 possible constructs. I did not have time to go through the detail of the 'X' construct. So, I am not sure why only these 4 constructs are listed. You have to read the original papers and find out why. At least, you now know how these 4 constructs are derived from the construct 'X'.

Ok, I found the orignal paper describing "confetti mice" (see attachment). The final products in figure 3B of the paper should be the real 4 constructs you should get. I don't know where you got the slide (in your question), where the A, B, C, and D constructs are just incompletely/partial deleted or inversed products. We have known that Cre is a very active recombinase enzyme, which should be able to perform 100% recombination-mediated deletion or inversion in mamallian and plant cells. The 4 constructs in figure 3B are the final products, generated through completely Cre-mediated deletion and inversion.

paper:

https://www.cell.com/cell/fulltext/S0092-8674(10)01064-0?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867410010640%3Fshowall%3Dtrue

Attachment: Figure 3B from the paper

Attached another figure will show you how the final 4 constructs are derived from the 'brainbow 2.1" construct (upon the expression of Cre recombinase)**.

**According to the paper, brainbow 2.1 construct was inserted at Rosa26 allele of the mice, and the mice later had an 'induciable' cre gene bred in. Upon induced, the cre gene expesses to produce Cre protein and recombine loxP sites on the brainbow 2.1 construct.

Let me know if you have more question on Cre-loxP site specific recombination system.

Dear Yuan-Yeu Yau ,

Thank you very much for your kind answers. I have seen these 2 papers before, but what´s not clear to me that if we obtain "Yellow" and "Blue" reporter as in the outcomes II and IV (attached picture 3B), double recombination events (removal of the stop cassette and inversion of GFP-YFP region) happen at the same time?

Also, I found on Jackson Lab website stated that: " “Initial Cre recombination outcomes may recombine the loxP-flanked STOP cassette (green), invert the loxP-STOP-loxP-GFP-PFY-Pxol region (yellow), recombine the loxP-STOP-loxP-GFP-PFY-Pxol-loxP region (red), or invert the entire loxP-STOP-loxP-GFP-PFY-Pxol-loxP-RFP-PFC-Pxol region (blue)” ( Mouse stock #017492 ). That´s why I wonder according to what they described, the stop cassette is still present if inversion events occur. For this, I draw the 4 constructs above (in my original question part on top) displaying what I understood from their statement, and I would like to ask you, the experts, if this model is correct in real.

Hi Mai Dan,

For your first question: if we obtain "Yellow" and "Blue" reporter as in the outcomes II and IV (attached picture 3B), double recombination events (removal of the stop cassette and inversion of GFP-YFP region) happen at the same time?

They are not necessary to happen at the same time. The recombination events could occur sequentially. For example: The first cassette is excised, then the second cassette is excised; finally, the remaining fragemnt between the two opposite loxP sites flip (inverse).

It could also happen this way: (1) the first cassette is excised (through loxP1 and loxP2 recombination). (2) Then, the new generated loxP1x2 site (through loxP1 and loxP2 recombination) can recombines with loxP5, and inverse the remaining DNA fragment between loxP1x2 and loxP5. (3) Then, loxP1x2 and loxP4 can recombine to remove 'green-and-yellow cassette', leading the generation of construct IV.

For your second question: "......Initial Cre recombination outcomes may recombine......":

Yes, 'Initial' Cre recombination might only result in partial DNA fragment excisions or inversions, but as long as the Cre proetin continueously present, it will keep working on loxP x loxP recombination until the 'cleanest' construct reaches (ex. construct I, II, III and IV) or until Cre activity is abolished (remember this Cre activity is induced).

FYI we're working with Brainbow 2.1 Confetti reporter in the blood right now, and we're seeing quite different expression of the four colors. Especially CFP is weak, because I think in the mouse it's expressed on the cell membrane, and as such there isn't much of it on there. We also only seeing around 20% of cells expressing Confetti for most mice. But overall the system works and produces different color combinations.

Daniel Whydoesthisrequirealastname : Thank you very much for sharing the info with me. I also found some other paper where they described differential expression of the 4 colors in various tissues, with GFP is less abundant.

Mai Dan Nguyen I've tested our spectral flow cytometer with proper controls and turns out it was confusing GFP and YFP.

So my conclusions have been changed. I observe that YFP and RFP are very abundant in the blood, while CFP and GFP are much less. The YFP/RFP/CFP/GFP ratio is roughly 10/9/2/1 across ~40 mice we have right now.