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Background The LoxP site based genetic switch, the FLEX, also known as DIO (Double-Floxed Inverted Open reading frame), was invented to turn on gene expression via Cre-mediated recombination. Since its first publication, numerous FLEX switch plasmids have been generated. These plasmids are designed to only work in combination with Cre. However on many occasions it is necessary to covert these FLEX plasmids back into constitutive expression plasmids so that they can also be used in non-Cre-expressing cells and in non-genetically modified animal models. Therefore developing a universal protocol for this purpose is useful as it could save a lot of valuable time and lab resources. Result Here we report a simple, quick, and cost-efficient protocol to invert the orientation of the open reading frame (ORF) within FLEX switch containing plasmids using commercial Cre recombinase. This protocol, requiring as little as 30 min and 50 ng of plasmid, has a cloning efficiency of 40–50%. To our surprise, single step recombination efficiency between the two mutant Lox2272 sites turned out very low. To understand this, we performed in vitro recombination assays. These assays revealed, significant impairment in recombination between Lox2272 sites as compared wild type LoxP sites in the FLEX plasmids. Conclusion We have presented an in vitro protocol to invert the ORF in FLEX based plasmids. This protocol is simple and highly efficient. Thus this method expends current molecular biology toolbox. We also demonstrate that the recombination between Lox2272 sites is much less efficient than wild type LoxP sites. This result has important implication for evaluating the efficacy of FLEX switch in biological systems and provides a rationale for future development of higher efficiency LoxP mutants.

A major stumbling block to understanding neural circuits is the extreme anatomical and functional diversity of interneurons. Subsets of interneurons can be targeted for manipulation using Cre mouse lines, but Cre expression is rarely confined to a single interneuron type. It is essential to have a strategy that further restricts labeling in Cre driver lines. We now describe an approach that combines Cre driver mice, recombinant adeno-associated virus, and rabies virus to produce sparse but binary labeling of select interneurons--frequently only a single cell in a large region. We used this approach to characterize the retinal amacrine and ganglion cell types in five GABAergic Cre mouse (Mus musculus) lines, and identified two new amacrine cell types: an asymmetric medium-field type and a wide-field type. We also labeled several wide-field amacrine cell types that have been previously identified based on morphology but whose connectivity and function had not been systematically studied due to lack of genetic markers. All Cre-expressing amacrine cells labeled with an antibody to GABA. Cre-expressing RGCs lacked GABA labeling and included classically defined as well as recently identified types. In addition to the retina, our technique leads to sparse labeling of neurons in the cortex, lateral geniculate nucleus, and superior colliculus, and can be used to express optogenetic tools such as channelrhodopsin and protein sensors such as GCaMP. The Cre drivers identified in this study provide genetic access to otherwise hard to access cell types for systematic analysis including anatomical characterization, physiological recording, optogenetic and/or chemical manipulation, and circuit mapping.