Off-Target Expression of Cre-Dependent Adeno-Associated Viruses in Wild-Type C57BL/6J Mice

Abstract

Adeno-associated viruses (AAVs) are a commonly used tool in neuroscience to efficiently label, trace, and/or manipulate neuronal populations. Highly specific targeting can be achieved through recombinase-dependent AAVs in combination with transgenic rodent lines that express Cre-recombinase in specific cell types. Visualization of viral expression is typically achieved through fluorescent reporter proteins (e.g., GFP or mCherry) packaged within the AAV genome. Although nonamplified fluorescence is usually sufficient to observe viral expression, immunohistochemical amplification of the fluorescent reporter is routinely used to improve viral visualization. In the present study, Cre-dependent AAVs were injected into the neocortex of wild-type C57BL/6J mice. While we observed weak but consistent nonamplified off-target double inverted open reading frame (DIO) expression in C57BL/6J mice, antibody amplification of the GFP or mCherry reporter revealed notable Cre-independent viral expression. Off-target expression of DIO constructs in wild-type C57BL/6J mice occurred independent of vendor, AAV serotype, or promoter. We also evaluated whether Cre-independent expression had functional effects via designer receptors exclusively activated by designer drugs (DREADDs). The DREADD agonist C21 (compound 21) had no effect on contextual fear conditioning or c-Fos expression in DIO-hM3Dq-mCherry+cells of C57BL/6J mice. Together, our results indicate that DIO constructs have off-target expression in wild-type subjects. Our findings are particularly important for the design of experiments featuring sensitive systems and/or quantitative measurements that could be negatively impacted by off-target expression.

Modified viral-genetic mapping reveals local and global connectivity relationships of ventral tegmental area dopamine cells

“…Off-target expression from Cre-dependent vectors has been reported previously. This problem was recently discussed in depth in several recent manuscripts ( Botterill et al, 2021 ; Fischer et al, 2019 ). The reasons underlying the off-target expression of transgenes are still unclear, though there are several possibilities.…”

“…The issue is not with the particular AAV vectors used for RABV mapping, but is representative of problems inherent in all standard AAV vectors. While FLEx loxP and DIO cassettes are typically sufficient to silence expression of fluorescent reporter genes and functional effectors such as DREADDs or optogenetic proteins, low levels of non-Cre-mediated, off-target expression are a problem when only a small amount of gene product is required, such as in the case of recombinase proteins or viral receptors ( Botterill et al, 2021 ; Miyamichi et al, 2013 ). In this case, even a low level of expression is sufficient to degrade specificity.…”

“…This leaky gene expression is due to incomplete suppression of transcription/translation of TVA. While this may not be an issue with fluorescent molecules or chemogenetic effectors such as DREADDs ( Botterill et al, 2021 ), in cases where only small amounts of a gene product are required to exert function, such as TVA, the problem becomes magnified. Only a single functional unit – presumably three TVA molecules bound to an EnvA trimer ( Alsteens et al, 2017 ) – is required to enable infection of EnvA-pseudotyped RABV.…”

Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo

“…Below, we discuss the most important insights rising from our work regarding use of viral tools and importance of careful attention to vector design as it can be used for targeting neuronal populations of different brain structures outside the olivocerebellar system (Gompf et al, 2015 ; Botterill et al, 2021 ).…”

The DREADDful Hurdles and Opportunities of the Chronic Chemogenetic Toolbox

“…Currently, there are 19 chemogenetic mouse lines commercially available at the Jackson Laboratory ( , accessed on 2 December 2021). Of note, the recombinase strategies can suffer from “leaky” expression, i.e., expression in the absence of the recombinase [ 48 ]. This is most certainly troublesome in transgenic mouse lines, in which the DREADD construct could have been inserted in the entire central nervous system and even in peripheral tissues, which makes it fundamentally difficult to exclude the effects of possible leaky expression on the study results.…”