Principle of Cre-DIO system

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Product list: Cre/loxP tools in AAV vector(AAV-Cre), adenoviral vector(Ad-Cre) and lentiviral vector(Lv-Cre)

Abstract

Cre-loxP system is widely used in the field of biosciences, especially in the generation of genetically engineered mice (knockout or overexpression), enabling researchers to study the function of gene of interest (GOI). To date, numerous systems, derived from the basic Cre-loxP system, have been developed to precisely control gene expression at desired time or cells. Here, we briefly introduce Cre-loxP system, and give a summary about its applications, especially viral vectors-mediated Cre expression (such as AAV-Cre or Ad-Cre) for in vitro or in vivo studies.

Principle of Cre-DIO

Although Cre-loxP tool is widespread in biological sciences, it is really restrictive to achieve more than one recombination event in a cell. Therefore, numerous loxP mutant versions have been created, with the variation from the unique 8 bp core asymmetric spacer “NNNTANNN”, such as loxN (GtATACcT), lox2272 (GgATACtT), lox5171 (ATGTGTaC), loxM2 (AgaaAcca), loxM3 (taaTACCA), loxM11 (AGATAGAA), and lox511 (GtATACAT). These lox variants only proceed recombination with the same type of lox sites, with no ability to interact with the other types. In Cre-DIO system, there are two pairs of lox sites (lox site 1 and lox site 2) flanking inverted GOI and reporter gene. In the absence of Cre, the inverted GOI and reporter are inactivated. Upon administration of Cre, the two pairs of lox sites will undergo recombination, resulting in the expression of GOI and reporter gene. Simultaneously, the cells with recombination interaction are labeled by the reporter gene (Fig. 6). The Cre-DIO tool utilizes initially inverted GOI to truly inactivate GOI, and activates GOI after a series of recombination interactions. Compared to the traditional overexpression strategy via floxed stop, this technology is much more effective to avoid the transcriptional leakage when the stop codon fails to totally suppress the transcription of GOI.

Figure 6. Principle of Cre-DIO (double-floxed inverse open reading frame) system. Without Cre, the GOI and reporter gene are inverted and silenced. In the presence of Cre, first, the transgene will be reoriented, then one of the two lox sites in each pair will be excised. Finally, the transgene contains only an incompatible pair of lox sites (lox 2-GOI-Reporter-lox 1) that are no longer responsive to Cre. Thus, the GOI and reporter gene will be activated. Lox site 1 and lox site 2 are variants of lox sites.

Summary

The Cre-loxP system, especially inducible tissue-specific knockout and viral vector-mediated Cre expression, has been highly utilized in genetics and cell biology research. Nevertheless, it seems that Cre-loxP system will continue to be prevalent in current and future research studies. GeneMedi is proficient in viral vector development and offers kinds of viral vector-based Cre tools, we can provide the best services and products if required.

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