27: Site-specific recombination
Summary
Both the Cre-lox and FLP-FRT systems are used for site-specific recombination to facilitate genome editing, especially for achieving tissue or temporal specificity.
Also known as
Variants: Cre-lox, FLP-FRT
Samples needed
Cells or organisms to be modified
Method
Site-specific combination requires two elements: A recombinase enzyme that catalyzes DNA cleavage and re-ligation, and two recombination sites in the DNA. The two most commonly used recombinase-recombination site pairs are Cre-lox and FLP-FRT. The recombination sites are made up of an asymmetrical core region where the cuts occur, flanked by inverted repeats where the recombinase binds the DNA. For recombination to occur between two recombination sites, the core sequences must be identical and they must be aligned in the same direction in space, which can require looping of the DNA. The directionality of the recombination sites is determined by the asymmetrical core sequence.
If the two recombination sites are aligned in the same direction in a piece of DNA, when recombination occurs, the intervening sequence will be removed. If the two sites are pointing toward one another, the intervening sequence will be inverted.
Site-specific recombination is often used in animal models. One of the benefits of site-specific recombination is that the genomic modification can be confined to a specific tissue by using a recombinase (Cre or FLP) under the control of a tissue-specific promoter. Alternatively, temporal specificity can be achieved through the use of inducible recombinases, like the Cre-ER fusion protein. Cre-ER only translocates to the nucleus, and therefore can only catalyze site-specific recombination, in the presence of either tamoxifen or 4-OHT, which can be supplied orally or injected. So, in this case, the researchers control the point at which the site-specific recombination occurs.
Controls
Unmodified control
Interpretation
In this experiment, researchers set out to identify all cells descended from cells expressing WOX5 , which is known to be expressed in the root primordia & root stem cell niche. To do this, they used an inducible Cre-lox system. As shown in “Construct 2” in panel B, this system used two reporters. Before recombination, GFP is expressed, and after recombination, GUS is expressed. GFP is fluorescent, and GUS is an enzyme that can cleave a chemical to produce a blue precipitate. This construct is under the control of the 35S ribosomal RNA promoter, which is active in all cells.
In this system, the researchers used a Cre fusion protein, Cre-GR (glucocorticoid receptor). This protein is usually found in the cytosol and will only enter the nucleus in the presence of the chemical dexamethasone (dex). Therefore, in order for site-specific recombination to occur, the Cre-GR protein must be expressed (only occurs in tissues where the WOX5 promoter is active) and dexamethasone must be provided by the researcher. Thus, the system provides both tissue specificity ( WOX5 promoter) and temporal specificity (dex administration). Once recombination occurs, the cell’s genome is permanently modified, and any descendents of that cell will also express GUS instead of GFP. In this way, one can identify all the descendants of particular cells. This kind of lineage tracing analysis is often used to identify all the descendants of particular stem cells.
In this experiment, the Zhai and Xu wanted to determine if the “adventitious” root cells, i.e. root cells that form from non-root tissue, like explanted leaves, are descendants of WOX5 -expressing cells. In panels E and F, we can see that the adventitious root (AR) cells and adventitious root primordium (ARP) both express GUS in the presence of dex, indicating that they do indeed descend from WOX5 -expressing cells. If their parent cell did not express WOX5 , they would not express Cre-GR, recombination would not have occurred, and the descendents would all express GFP instead of GUS.
Image Descriptions
Figure 1 image description:
A: Photo of a leaf with an adventitious root (AR) growing from it.
B: Schematic of Cre-lox system. Construct 1: WOX5 pro - Cre-GR - ter. Transcription leads to Cre-GR protein. Construct 2: S35 pro - LOX - GFP - ter - LOX - GUS - ter. Cre-GR + dex leads to recombination to: S35 pro - GUS - ter , which leads to GUS signal.
C: Photo of seedling showing green fluorescence.
D: Photo of root showing green fluorescence.
E: Photo of two explanted leaves with ARs growing from them. Left, +dex, shows dark blue staining throughout AR, whereas right, -dex, shows no staining.
F: Photo zooming in on bottom of explanted leaf. There is a spot of GUS staining near the bottom of the leaf labeled ARP for adventitious root primordium.
Thumbnail
"Cre-lox inversion.svg" ↗ by Staticd is licensed under CC BY-SA 3.0 ↗.
Description: Inversion of a DNA sequence between two inverted Lox sites.
Author
Katherine Mattaini, Tufts University
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Zhai, N., and L. Xu. 2020. CRE/LOX-based analysis of cell lineage during root formation and regeneration in Arabidopsis. aBIOTECH 1:153–156. ↵