Detecting and Avoiding Problems When Using the Cre–lox System

Abstract

The Cre-lox recombination approach is commonly used to generate cell-specific gene inactivation (or activation). We have noticed that the breeding and genotyping sections of papers utilizing Cre-lox techniques are frequently incomplete. While seemingly straightforward, there are important considerations that need to be implemented in the breeding and genotyping methods to prevent the introduction of experimental confounds. Germline recombination and transient expression of Cre recombinase during development are some examples of the complications that can occur, and conventional genotyping methods may fail to identify these events. In this opinion article, we highlight the importance of testing for unexpected recombination events, suggest strategies to isolate and minimize adverse recombination events, and encourage editors and reviewers to expect more definitive statements regarding the validation of genotyping.

Mouse Cre Models for the Study of Bone Diseases

“…It should be done by a combination of PCR on the genomic DNA to detect the recombined DNA sequence and qPCR or western blotting to confirm whether mRNA or protein expression levels of the target gene are altered in targeted and non-targeted tissues. Additional information on how to evaluate Cre transgenic lines and avoid problems when using them is provided in an excellent review by Song and Palmiter [9]. While off-target Cre expression does not necessarily negate the results of conditional knockout experiments, it must factor into a more nuanced interpretation of the data.…”

“…The conditional gene inactivation or activation approach exploits the Cre-loxP system in which Cre recombinase (Cre) is used to excise or invert “target” DNA sequences that have been engineered to be flanked by two 34-bp DNA sequences, termed loxP sites (reviewed in [9, 10]). Cre recombinase is a 38kDa protein originally discovered in bacteriophage P1 [11] that mediates site-specific intermolecular DNA recombination between two loxP sites.…”

Craniofacial skeletal response to encephalization: How do we know what we think we know?

“…Recent advancement of this site-specific recombinase technology enables specific cell types or developmental time points to be precisely targeted or a specific external stimulus to act as a trigger for the Cre-recombinase activity. Though some Cre lines have been found to not always behave as expected (see, e.g., Davey et al, 2012; Matthaei, 2007; Schmidt-Supprian & Rajewsky, 2007; Smith, 2011; Song & Palmiter, 2018), tissue- and cell-specific Cre-recombinase lines offer the potential to control the expression pattern of any transgene in anatomical space and/or developmental time. Many cre drivers that target aspects of neurogenesis are available (Table 2).…”

Brain architecture at varying scales

“…For optimal results, each of these levels requires a specific visualization technique. Fortunately, recent advances in technology, including more sensitive microscopes with faster scanning speeds and robotics, novel molecular tools, labeling methods, sophisticated software, data repositories and computational models, are rapidly increasing our knowledge of brain circuits (advanced imaging: (Liu et al, ; Sahl, Hell, & Jakobs, ) and whole‐brain imaging (Economo et al, ); Cre‐lox system in neuronal tissue (Song & Palmiter, ; Tsien et al, ): for a comprehensive review on clarity: (Richardson & Lichtman ); emerging labeling tools: (Bedbrook, Deverman & Gradinaru, ; Luo, Callaway, & Svoboda, ; Sarno & Robison, )). This In Focus issue was inspired by the multilevel resolution imaging challenge on one hand, and contemporary tool innovation on the other, both assisting in bringing these varying levels of resolution closer together to better understand the functional organization of the brain (Figure ).…”