Some Basics about Cre-lox and Inducible Cre in Mouse Modeling

Cre-lox system, characterized by simple manipulation and high-efficiency recombinase, has been employed as a powerful tool of gene-editing. Application of Cre-lox system can achieve time-specific knock-out and knock-in of target genes in specified cells, tissues, and organisms, thereby regulating genes of interest spatiotemporally, which has profound impact on research of genetic function and animal modeling.

What is Cre-lox system?

Cre-lox is composed of a recombinase and a pair of short target sequences, Lox sequences.

Cre recombinase

Cre recombinase, one of tyrosine recombinase enzymes, can catalyze site-specific DNA recombination between specific sites in a DNA molecule. Derived from bacteriophage P1, Cre recombinase consists of 343 amino acids, and can specifically bind to loxP sites. Besides, flipase (FLP) and D6 specific recombinase (Dre) bear a resemblance in action and usage to Cre recombinase.

Lox sites

Lox sites, also designated as loxP sites(locus of X-over P1 sites), is recognized by Cre recombinase, and composed of 34 bp characteristic sequences, ATAACTTCGTATA -NNNTANNN-TATACGAAGTTAT. The 13 bp on both sides are palindromic, and recognition sites of Cre recombinase, while 8 bp spacer provides site for recombination, and determines the direction of loxP sites. N indicates the variable base, which may cause different loxP sites. Apart from wild type loxP site, there exist some other widespread loxP sites, such as Lox2272, Lox511, and Lox5171, which can also be recognized by Cre recombinase.

                                                           Wild-type LoxP site and its permutations 

There exist three types of recombination events, depending on the location and the orientation of lox sites in a DNA molecule.

Deletion:If the two loxP sites are in the same orientation, the floxed sequence (sequence

flanked by two loxP sites) is excised;

Inversion:if the two loxP sites are in the opposite orientation, the floxed sequence is inverted;

Translocation:If there exists a floxed donor sequence, the donor sequence can be swapped with the original sequence.

Figure 2 Cre recombination mechanism. A. Structure of loxP sites, the orientation of which is indicated by red arrow; B. Cre recombinase-mediated cassette exchange; C. Cre recombinase-mediated inversion and excision.

Conditional gene targeting in mice: In vivo applications of Cre-lox system

In principle, two transgenic mouse models (providing two different genetic components, respectively) will be required to achieve time specific knock-out, knock-in, and activation of target genes in mice.

The first step is to establish Cre-expressing mouse strain. The cre recombinase driven by specific promoters can function in specific cells, tissues and whole body. Cre expression can be controlled using an inducible promoter or regulator. 

The second step is to establish Flox mouse strain by flanking the target gene using lox sites. And the Cre-lox expressing strain(Conditional gene targeting strain) will be achieved by crossing the two mouse strains.  

Conditional gene activation

Cre-lox system was initially utilized for cell type-specific gene activation in mice. Generally speaking, the mouse models applied to conditional gene activation require the insertion of a silencing sequence, flanked by lox sites between target gene and promoter, thereby designated as “Lox-Stop-Lox” (LSL) stop cassette.The stop cassette is commonly composed of polyA and splice-donor sequences, and able to hinder downstream gene transcription. When the flox mouse strain is crossed with cell type-specific Cre-expressing mouse strain, the F1 mouse strain will generate stop cassette-deleted Cre-expressing cells, which result in the activation of target gene.  

             Figure 3 Mechanism of conditional gene activation

Conditional gene knock-out

Flox mouse strain, designated to conditional gene knock-out, requires the flanking of target gene using lox sites with the same orientation(lox-GENE-lox). When flox mouse strain is crossed with cell type-specific Cre-expressing mouse strain, Cre recombinase will recognize lox sites, which results in the deletion of target gene. Since Cre expression is regulated by upstream promoter, spatiotemporal specificity of promoter elicits that of gene recombination.

       Figure 4 Mechanism of conditional gene knock-out in mice

Inducible Cre-lox system

Time-specific Cre-lox system, also referred to as inducible Cre-lox system, is intended for genetics research with higher accuracy, and has been widely applied to development stage-specific gene function research and strain tracking. There exist several types of commonly used inducible Cre-lox system.

CreER-lox system

CreER-lox system, also designated as Tamxifen (Tam)-inducible Cre system, can achieve Tam-induced recombination through the fused protein of Cre and ER. In the absence of Tam, fused protein CreER, initially localized in cytosol, binds to HSP90. However, the presence of Tam will disrupt the interaction between CreER and HSP90. Meanwhile, the interplay between ER and TAM will facilitate nuclear translocation of Cre. In nucleus, CreER can recognize loxP sites, and elicit the inactivation of gene X in tissue Y. CreER2 always presents higher sensitivity to drug induction in vivo and superiority in usage.

                                          Tam-inducible Cre system

Tetracycline (Tet) system

Tetracycline (Tet) system, also called doxycycline (Dox; a tetracycline derivative)-inducible Cre system. There exist two modes for this system, Tet-on and Tet-off, which permit Dox-dependent gene activation and inactivation. Tet system consists of reverse tetracycline-controlled transactivator (rtTA), tetracycline-controlled transactivator (tTA) and tetracycline responsive element (TRE), also referred to as a tetracycline operon (TetO), which regulates a cre gene expression. In Tet system, doxycycline is usually administered in feed or drinking water.

Tet-on system

In Tet-on system, there exist ubiquitous rtTA and tissue-specific promoter-driven rtTA. In absence of DOX, the inactivated rtTA is unable to bind to TRE sequence, which is responsible for Cre regulation, so as to disable Cre expression. However, after the administration of Dox, Dox can bind to rtTA and activate it, which further result in the binding between activated rtTA and Tre sequence, and then the induction of Cre expression.

                            Dox-inducible cre system; Tet-on system.

Tet-off system

In Tet-off system, in absence of Dox, activated tTA can bind to upstream TRE sequence of Cre and induce Cre expression. However, in the presence of Dox, tTA is inactivated, and its binding to TRE is disrupted, which then inhibits Cre expression.

                                       Dox-inducible cre system; Tet-off system.