Researchers at University of Tsukuba have developed a vector that integrates the Cre enzyme gene with the specific DNA sequence loxP, which is its target. This vector allows to create Cre-loxP organisms, which are widely used in the functional analysis of specific genes, in a single step and thus to significantly reduce the time and cost of production.

Tsukuba, Japan—Organisms modified via the introduction of the Cre-loxP system (Cre-loxP organisms) are widely used in life sciences to investigate the function of specific genes. Cre is a type of DNA recombination enzyme that binds to a short DNA sequence (loxP) of 34 base pairs and causes site-specific recombination. For example, by inserting a DNA sequence between two loxPs, Cre can remove the DNA sequence (knock out). Furthermore, by changing the orientation and arrangement of loxP, it is also possible to invert the orientation of specific DNA sequences or transfer them to other locations in the genome.

Cre-loxP organisms are generally produced by separately creating Cre-lineage and loxP-lineage individuals that contain the Cre gene and loxP in their genome, respectively, and then crossing them. This process is costly and time-consuming. However, developing a vector containing both the Cre gene and loxP (a Cre-loxP integrated vector) for introduction into the genome of fertilized eggs would allow to produce Cre-loxP organisms in a single step. This would eliminate the need for crossbreeding and would result in significant reductions in the time and cost of production. However, this has proved challenging to achieve because the Cre gene in the vector is spontaneously expressed in the E. coli bacteria used to synthesize such vector, causing recombination to occur in the loxP region within it.

In this study, the researchers succeeded in synthesizing a Cre-loxP integrated vector by placing a short DNA sequence called TAx9 before the promoter region of the Cre gene, thereby preventing its expression in E. coli. They also demonstrated that this technology can be applied to newts and mice. While continuing their research on newt regeneration, this research team accidentally succeeded in creating a Cre-loxP integrated vector. The TAx9 technology was born in the process of elucidating its mechanism and it is expected to be applied in various life science fields. The University of Tsukuba is currently applying for a patent for this technology.

###
This research was supported by the TMRC Collaborative Research Program (TB22-04; TB23-01) and the Japan Society for the Promotion of Science (24240062; 18H04061; 23H05483) to C.C.

Original Paper

Title of original paper:

One-step Cre-loxP organism creation by TAx9

Journal:

Communications Biology

DOI:

10.1038/s42003-025-07759-9

Correspondence

Professor CHIBA Chikafumi
Assistant Professor Martin Miguel Casco-Robles
Institute of Life and Environmental Sciences, University of Tsukuba

Related Link

Institute of Life and Environmental Sciences