With the advantages of simple operation and high recombination rate, the Cre-Lox system has now become a powerful tool for genetic manipulation across in vivo and in vitro. The Cre-Lox system enables gene expression with exceptional spatial (expresses a gene in specific cells, tissues, or entire organisms) and temporal (at specific time points through the application of the Cre-Lox system) control. Application of the Cre-Lox system is often used to achieve custom spatiotemporal operations on specific genes, which is useful for the study of gene function and animal models of human disease. Herein, we explore the Cre-Lox system and its profound impact on animal model construction capabilities.

 

1.  What is the Cre-Lox System?

There are two main parts of the Cre-Lox system, both of which have been adapted from the P1 bacteriophage:

 

Cre Recombinase

Cyclization recombinase (Cre) is one of the tyrosine site-specific recombinases, which is known to catalyse the site specific recombination event between two DNA recognition sites (LoxP sites). Cre recombinase consists of 343 amino acids, which can specifically recognize Lox sites. In addition to Cre, there are flipase (Flp) and D6 specific recombinase (Dre) – although these enzymes still demonstrate a lower recombination efficiency compared with Cre.

 

LoxP Sites

The palindromic DNA region recognized by Cre recombinase are known loxP (locus of X-over P1) sites. LoxP sites are directional 34 bp sequences made up of two 13 bp reverse complement on both sides (the recognition sequence of Cre recombinase) and an 8 bp spacer region (the position where recombination occurs) which gives it directionality. The orientation of LoxP sites can result in three genetic recombination events, including excision, inversion, and translocation. In addition to wild type LoxP sites, there are common sites such as Lox2272, Lox511, Lox5171, and more - these mutant Lox sites can also be recognized by Cre recombinase. However, only two of the same mutant lox sites can recombine with each other.

 

Table 1. Published mutant loxP sites

loxP site Left inverted repeat sequence Spacer (5'→3') Right inverted repeat sequence
Wild-type ATAACTTCGTATA ATGTATGC TATACGAAGTTAT
lox 511 [6] ATAACTTCGTATA ATGTATaC TATACGAAGTTAT
lox 5171 [7] ATAACTTCGTATA ATGTgTaC TATACGAAGTTAT
lox 2272 [7] ATAACTTCGTATA AaGTATcC TATACGAAGTTAT
m2 [8] ATAACTTCGTATA AgaaAcca TATACGAAGTTAT
m3 [8] ATAACTTCGTATA taaTAcca TATACGAAGTTAT
m7 [8] ATAACTTCGTATA AgaTAgaa TATACGAAGTTAT
m11 [8] ATAACTTCGTATA cgaTAcca TATACGAAGTTAT
lox 71 [12, 13] taccgTTCGTATA ATGTATGC TATACGAAGTTAT
lox 66 [12, 13] ATAACTTCGTATA ATGTATGC TATACGAAcggta

 

 

2. Development of Conditional Models Using Cre-Lox

Compared with constitutively expressed gene mutations, conditional models provide greater temporal and regional control of gene expression. The Cre-Lox recombination system enables the generation of tissue-specific or inducible knockouts with a high level of control over the spatial and temporal expression of genes.

 

Figure 1. Mechanism of Cre-LoxP System

 Mechanism of Cre-LoxP System | Cyagen

(A) An overview of Cre-LoxP system. 38 kDa Cre recombinase recognizes the loxP sites of specific 34 bp DNA sequences, excising the sequence and inactivating Gene Y. (B) General breeding strategy for conditional mutation using loxP and Cre driving mouse line. Tissue-specific Cre-driver crossing with Floxed alleles of gene Y leads to inactivation of Gene Y in tissues associated with Promoter X.

 

CRISPR Capabilities for Mouse and Rat Models:

As outlined in Figure 1, the basic Cre-loxP recombination event is most often used for controlled excision of genetic sequences. This has been adapted to create Cre-dependent sequence knockout (KO) – depicted in figure 1 - and Cre-dependent gene expression models – such as the transgene switch strategies described below.

 

  • Standard Use - Generate tissue-specific or inducible knockouts(e.g. Cre-dependent sequence KO), such as in the KO-first conditional allele strategy
  • Transgene Switch – (Cre-dependent gene expression)
  1. Transgene Activation: A loxP-flanked “stop” sequence between the promoter and the coding sequence blocks expression (until bred with a Cre strain)
  2. Inducible Knockout(KO): A loxP-flanked transgene sequence may be removed upon breeding with a Cre strain.
  • Reporter Strains - Visually confirm locations of Cre recombinase activity with loxP-flanked fluorescent protein sequences

 

Cyagen Cre Model Platform

Despite the utility of the Cre-Lox modeling strategy, the complexity of breeding protocol alongside the increased cost of rat colony maintenance has created significant barriers for researchers seeking to make novel conditional rat models. Another major constraint faced by researchers developing cKO or cKI rat models is the lack of Cre lines specific to the target of interest.

 

As a leader in custom rodent models, Cyagen is building a repository of Cre loxP model resources, including Cre driver rat strains. We are offering a complete range of custom conditional knockout (cKO) or conditional knockin (cKI) rat models alongside the Cre driver strains needed to develop physiologically relevant models of human diseases, available to researchers worldwide.

 

Some of the current Cre rat strains under-development:

alb (Cre Ert2) myh6 (Cre Ert2) slc6a3 (Cre Ert2) camk2a (Cre Ert2) nphs2 (Cre Ert2)
alb (Cre) myh6 (Cre) slc6a3 (Cre) camk2a (Cre) nphs2 (Cre)
gfap (Cre) gfap (Cre Ert2) nes (Cre) nes (Cre Ert2)  

Still looking for your Cre line? Cyagen can provide pricing accommodations to researchers interested in Cre lines not currently available in our listings. Contact us to inquire about your conditional model needs and see how we can help develop a custom Cre strain to support your field of study.

 

For a limited time, order any cKO/cKI rat models to receive a free Cre driver rat strain custom-developed to support your field of study. Place an order by November 30, 2020 to qualify for your free Cre strain.

 

 

About Cyagen

Cyagen offers a "one-stop shop" tailored to your gene research and model generation needs. Our services range from DNA vector construction to embryonic stem cell manipulation, microinjection, and breeding. All projects are fully customizable and flexible.

 

  • AAALAC-accredited and OLAW assured animal centers
  • Specific-pathogen-free (SPF) animal health status exceeds requirements of most facilities
  • Over 50,400 mouse and rat models delivered worldwide
  • Cited in over 4,000 publications across high-impact journals (Nature, Immunity, etc.)
  • Top-tier customer support: free consultations and quotes
  • 100% money-back guarantee

 Cre Loxp Mice - Photos of the Cyagen Transgenic Animal Center (CTAC)

Photos of the Cyagen Transgenic Animal Center (CTAC)

 

References:

  1. Kim H, Kim M, Im SK, Fang S. Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes. Lab Anim Res. 2018;34(4):147-159. DOI:10.5625/lar.2018.34.4.147
  2. Missirlis PI, Smailus DE, Holt RA. A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination. BMC Genomics. 2006;7:73. Published 2006 Apr 4. DOI:10.1186/1471-2164-7-73

 

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