A Guide to Creating and Testing New INTRSECT Constructs

Abstract

As the power of genetically encoded interventional and observational tools for neuroscience expands, the boundaries of experimental design are increasingly defined by limits in selectively expressing these tools in relevant cell types. Single-recombinase-dependent expression systems have been widely used as a means to restrict gene expression based on single features by combining recombinase-dependent viruses with recombinase-expressing transgenic animals. This protocol details how to create INTRSECT constructs and use multiple recombinases to achieve targeting of a desired gene to subsets of neurons that are defined by multiple genetic and/or topological features. This method includes the design and utilization of both viruses and transgenic animals: these tools are inherently flexible and modular and may be used in different combinations to achieve the desired gene expression pattern. © 2017 by John Wiley & Sons, Inc.

Endocannabinoid Signaling Collapse Mediates Stress-Induced Amygdalo-Cortical Strengthening

“…However, plPFC-specific DAGLα deletion could exert behavioral effects via impairment in 2-AG signaling at other limbic glutamatergic inputs or local GABAergic terminals. To further solidify the role of 2-AG-CB1 signaling specifically at BLA-plPFC synapses in the regulation of stress-induced anxiety, we utilized an INTRSECT approach to selectively delete the CB1 receptor from BLA neurons projecting to the plPFC (Fenno et al, 2017). Using a mouse in which the single coding exon for the CB1 receptor is flanked by loxP sites (Figure S6a-e), we injected a retrograde virus that drives the expression of Flp recombinase and td-Tomato into the plPFC and a virus that drives expression of Cre recombinase in a Flpdependent manner into the BLA.…”

Widespread inhibitory projections from the cerebellar interposed nucleus

“…Control injections were performed where Cre or Flp expression was occluded, either by performing the injections in wild type mice or in transgenic mice without the Retro-flp injection into IO or RN, confirming the necessity of recombinase presence in reporter expression (Fenno et al, 2017). Kim et al, 2016;Wall et al, 2010;Wickersham et al, 2007).…”

“…Several important questions and caveats remained, however, including that Purkinje neuron label was unavoidable with direct injection into Gad1-Cre and Vgat-Cre IntA. We next used an intersectional approach to restrict label to IOprojecting iIntA neurons (Fenno et al, 2017). In Gad1-cre (n=4) and Vgat-cre mice (n=3), we injected the contralateral IO with AAVretro-EF1a-Flp followed by a two-recombinase-dependent reporter virus (AAV8-hsyn-ConFon-eYFP) into IntA (Figure 2A-B Figure 2D, G), even in the most finely targeted injections.…”

Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation

“…Inclusion of an intron can have positive impacts on expression levels. Introns have also been combined creatively with recombinase sites and partially inverted transgenes to achieve tight intersectional control of transgene expression (Fenno et al, 2014, 2017). Many recombinant AAV genomes also include a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), which can dramatically enhance expression.…”