Cre-lox and Other Recombinase Systems: An Overview
Cre-lox and other recombinase systems are among some of the most versatile and impactful genetic tools in recent decades. These tools enable precise control over the timing and localization of gene expression. This article briefly explores how these systems work and their use in mouse models.
The Cre-lox system
Introduced in the 1980s and patented by DuPont Pharmaceuticals, Cre-lox technology has been effectively used to modify genomes in plants, insects, fish, and mammals including mice. This technology utilizes the recombinase Cre, derived from a P1 bacteriophage, to induce recombination at specific lox sites (lox sites include the commonly-used loxP, as well as lox2272 and loxN among others), allowing for the activation or inactivation of target genes in mice.
To utilize Cre-lox technology, researchers typically create a Cre-lox mouse by breeding a Cre-expressing mouse with a lox-containing mouse. The Cre mouse carries a Cre recombinase transgene under the control of a tissue-specific promoter, while the lox mouse contains two lox sites flanking a genomic segment of interest, known as the "floxed" locus. These mice are typically produced using transgenic technology.
Cre mice can be engineered to express Cre recombinase under specific conditions based on the promoters and regulatory controls used. These conditions may include expression in particular tissues, in response to dietary supplements like doxycycline, tetracycline, RU486, and tamoxifen, or during specific developmental stages.
The orientation and location of lox sites also influence Cre-lox reactions. Paired lox sites have directionality and can be arranged either in cis (on the same DNA strand) or in trans (on different DNA strands).
Excision or Circularization: When lox sites flank a DNA segment in a cis arrangement and are oriented in the same direction, Cre recombinase facilitates the excision or circularization of the segment.
Inversion: If the lox sites are in a cis arrangement but oriented in opposite directions, Cre recombinase mediates the inversion of the segment.
Translocation: When lox sites are located on different DNA strands and oriented in the same direction, Cre recombinase mediates the translocation of the segment.
Therefore, depending on the location and orientation of the lox sites within the Cre-lox mouse genome, Cre recombinase can facilitate deletions, inversions, and translocations of a floxed locus.
Typically, Cre and lox strains are developed independently and then crossed. Numerous Cre strains, each containing a Cre transgene driven by a different tissue-specific promoter, can be crossed with a single lox strain. Depending on the strains mated, a variety of Cre-mediated model systems can be constructed, including transgenics, knockouts, hypomorphs, repairable hypomorphs, chromosome aberrants, and diet-induced mutants. By mixing and matching Cre and lox strains, researchers can study gene effects in tissue-specific and developmental stage-specific contexts that were previously unattainable.
Alternative Recombinase Systems
While the Cre-lox system has proven invaluable, the genetic toolbox has expanded to include other alternative recombinase systems. Among these are Flp, Dre, and Cre-ERT, each operating on a similar conceptual framework to the Cre-lox system. Except for the Tet system (which manipulates gene expression but does not use a recombinase), they all involve a recombinase that targets sequences flanking a specific genomic region. This strategic flanking can facilitate the excision, inversion, translocation, or expression of genetic material, allowing for precise genetic manipulation with spatial and temporal control.
Recombinase systems can be broadly categorized into two types: constitutive and inducible systems. In a constitutive recombinase system, the target gene's expression is constant, while in inducible expression, gene expression is induced under certain conditions only, such as from the addition of a dietary supplement.
Constitutive Systems
FLP-frt
The Flp-frt system employs the serine recombinase FLP, derived from the yeast S. cerevisiae, to recognize pairs of FLP recombinase target (frt) sequences that flank a specific genomic region. frt sites consist of three 13 bp reverse palindromes and an eight bp spacer sequence, totaling 48 bp. Compared to the Cre-lox system, Flp-frt has lower efficiency for recombination events. Additionally, FLP recombinases are temperature-sensitive, with optimal activity at 30°C. Activity significantly decreases above 30°C, with little detectable activity past 39°C, making Flp less suitable for mammalian cells. Although thermostable variants, FLPe and FLPo, have been identified, it still exhibits lower efficiency than Cre recombinase.
Dre-rox
The Dre-rox system operates efficiently in mice and utilizes the tyrosine recombinase Dre derived from the D6 bacteriophage. Dre recognizes the rox sequence, which consists of two 14 bp reverse palindromes and a 4 bp intermediate spacer. Dre shares significant homology with Cre, with the rox site differing by only 3 out of 13 nucleotides per half-site. Despite this homology, cross-recombination between the two systems is generally absent, although some groups have reported slight cross-reactivity under conditions of high recombinase expression.
Inducible Systems
CreERT-lox
This system uses Cre technology with a tamoxifen-inducible Cre-estrogen receptor (ERT) fusion protein. Normally sequestered in the cytoplasm, the CreERT proteins translocate to the nucleus upon tamoxifen introduction, where they facilitate recombination at loxP sites. This inducibility allows researchers to control genetic recombination events with precise timing, enabling the activation or deactivation of target genes with temporal control as needed.
Tet-on and Tet-Off
The Tet-on and Tet-off systems are complementary methods used to regulate the expression of a target transgene through an inducible transcriptional activator. These systems utilize varying concentrations of transcriptional activators, typically the antibiotics tetracycline (Tc) or its derivative doxycycline (Dox), to reversibly and quantitatively control transgene expression.
In the Tet-off system, a tetracycline-controlled transactivator protein (tTA) regulates the expression of a target transgene under the control of a tetracycline-responsive promoter element (TRE). Without Tc or another transcriptional activator, tTA binds to the TRE and activates the transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, resulting in the inactivation of the target gene's expression.
Meanwhile, the Tet-on system utilizes a reverse tetracycline-controlled transactivator (rtTA). A small four amino acid change in the rtTA protein renders the protein only able to recognize the TRE sequence of a target transgene in the presence of a Dox effector. As a result, Dox induces the transcription of the TRE-regulated transgene in the Tet-on system.
Intersectional Genetics
With a range of recombinase systems available, a distinct advantage arises in enhancing our capability to spatially and temporally manipulate genomes beyond the capabilities of a single Cre-lox or other recombinase system. This innovative approach, termed intersectional genetics, strategically leverages multiple recombinase systems to achieve a level of precision that is unattainable by any individual system. It enables highly targeted manipulation of gene expression, including precise control of reporter gene expression, gene knockouts, and knock-ins, all within a single model. For more about intersectional genetics, please read more in this blogpost.