The Cre-loxP

  The Cre-loxP system is a widely-used tool in genetic engineering that provides researchers with precise control over gene expression and modification. This system enables targeted deletion, insertion, or rearrangement of specific DNA sequences, making it a powerful instrument for studying gene function and developing therapies for genetic diseases.

At the core of the Cre-loxP system is the Cre recombinase enzyme, which can identify and cut DNA at specific loxP sites. These loxP sites are small DNA sequences introduced into specific locations in a DNA sequence, allowing researchers to manipulate the sequence as needed. One significant advantage of the Cre-loxP system is its versatility. It enables the creation of tissue-specific knockout models, which can help investigate gene function in specific cell types or tissues. Additionally, this system can create temporal knockouts, allowing researchers to study gene function at different developmental stages or in response to specific stimuli. The Cre-loxP system has revolutionized the field of genetic engineering, providing researchers with a powerful tool for studying gene function and developing new therapies for genetic diseases.

Cre recombinase is a type of tyrosine site-specific recombinase, also known as T-SSRs. This enzyme is approximately 38-kDa in size and is coded by the cre (cyclization recombinase) gene of bacteriophage P1. The function of Cre recombinase is to recognize and target specific DNA sequences known as loxP (locus of x-over, P1) sites. A loxP site is a 34-base-pair DNA sequence that consists of two 13-base-pair inverted and palindromic repeats, as well as an 8-base-pair core sequence.

Figure 1: Lox P site 

The Cre-loxP system involves the use of Cre recombinase enzyme that recognizes and binds to two directly repeated loxP sites. Upon binding, the Cre enzyme excises the loxP flanked (floxed) DNA, resulting in two types of DNA with circular, excised, and inactivated gene.

In addition to deletion, the Cre-loxP system can also induce inversion and translocation of DNA between two loxP sites (Figure 2). The orientation and location of loxP sites determine the direction and outcome of the DNA manipulation. The Cre-loxP system offers precise control over gene modification and is a widely used tool in genetic engineering.

Figure 2: LoxP orientation and cre recombinase  effect 

The Cre recombinase can be used for conditional gene manipulation, which typically involves two steps.

Firstly, a Cre- strain is generated, where the Cre recombinase is controlled by a promoter that targets specific cells or tissues of interest. This allows for precise spatial and temporal control over the activation of Cre recombinase.

Secondly, a mouse strain is generated that contains DNA sequences flanked by loxP sites (floxed DNA). These loxP sites serve as recombination targets for the Cre recombinase, which enables the precise deletion, inversion, or translocation of the floxed DNA sequence in the cells or tissues where the Cre recombinase is active.

Using the Cre and floxed DNA strains, conditional gene manipulation is possible, and the investigation of gene function can be carried out in specific cell types or at specific developmental stages.

Figure 3: Construction of Conditional knockout mouse 

The Tamoxifen-inducible Cre system is a genetic engineering technique that enables scientists to control gene expression in specific cells at specific times (Figure 3). 

The method uses a modified Cre protein called CreER recombinase, which is fused with an estrogen receptor that has a mutated ligand binding domain (ER-LBD). 

When Tamoxifen or 4-hydroxytamoxifen (4-OHT) is administered, it disrupts the interaction between CreER and heat shock protein 90 (HSP90).

 As a result, the CreER recombinase translocates to the nucleus and interacts with specific DNA sequences called loxP sites to modify gene expression. 

 system is regulated by cell-specific regulatory elements like promoters and enhancers. The inducer can be administered at a specific time to induce Cre activity in the desired cells. 

This method allows for more precise control of genetic modifications and has a wide range of applications in research on gene function and the development of new therapies.

Figure 3: Tamoxifen-inducible Cre system 

 

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References:

•       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 Dec;34(4):147-159. doi: 10.5625/lar.2018.34.4.147. Epub 2018 Dec 31. PMID: 30671100; PMCID: PMC6333611.