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Review
. 2015 Jul 1:72:1.24.1-1.24.15.
doi: 10.1002/0471142301.ns0124s72.

Cre Activated and Inactivated Recombinant Adeno-Associated Viral Vectors for Neuronal Anatomical Tracing or Activity Manipulation

Arpiar Saunders  1 , Bernardo L Sabatini  1
Affiliations
Review

Cre Activated and Inactivated Recombinant Adeno-Associated Viral Vectors for Neuronal Anatomical Tracing or Activity Manipulation

Arpiar Saunders et al. Curr Protoc Neurosci. .

Abstract

Recombinant adeno-associated viruses (rAAVs) transcriptionally activated by Cre recombinase (Cre-On) are powerful tools for determining the anatomy and function of genetically defined neuronal types in transgenic Cre driver mice. Here we describe how rAAVs transcriptionally inactivated by Cre (Cre-Off) can be used in conjunction with Cre-On rAAVs or genomic Cre-reporter alleles to study brain circuits. Intracranial injection of Cre-On/Cre-Off rAAVs into spatially intermingled Cre(+) and Cre(-) neurons allows these populations to be differentially labeled or manipulated within individual animals. This comparison helps define the unique properties of Cre(+) neurons, highlighting the specialized role they play in their constituent brain circuits. This protocol touches on the conceptual and experimental background of Cre-Off rAAV systems, including caveats and methods of validation.

Keywords: AAV; Cre-dependent virus; Cre-lox; rAAV; viral tracer.

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Figures

Figure 1
Figure 1
rAAV genome organization and manipulation by Cre through incompatible lox sites. A. Top, rAAV particles contain a single stranded (ss) DNA genome in which the endogenous rep and cap genes are replaced by an expression cassette containing a promoter, transgene and polyadenylation signal. Lox sites situated within this cassette confer Cre-sensitive expression. The only required endogenous rAAV sequences are the inverted terminal repeats (ITRs), which self-prime in the nucleus to polymerize into a double-stranded (ds) form. Bottom, ds-rAAV genomes can remain linear (i) or circularize, remaining independent (ii) or undergo ITR-mediated recombination into multimeric concatamers (iii). Cre-mediated recombination may also produce other forms of multimeric ds-rAAV genomes. B. Sequence comparison of recombination incompatible lox sites used in Cre-On and Cre-Off rAAVs. Lox sites (34bp) contain two 13bp palindromic recognition sequences and an 8bp non-palindromic spacer region that confers directionality and compatibility to recombination. Recombination occurs between lox site pairs in two basic steps. First, two Cre molecules bind each of the recognition sequences on a single lox site. Second, two pairs of Cre-bound lox sites form a complex, catalyzing DNA synapsis and subsequent strand exchange within the spacer region. The efficiency of strand exchange depends on spacer region sequence similarity. LoxP, lox2272 and loxFAS recombine efficiently with like-pairs but not unlike-pairs (Siegel et al., 2001). Differences in the lox sequences are highlighted.
Figure 2
Figure 2
Cre-On/Off transgene expression in DIO, DO and FAS rAAVs. A. Oppositely oriented loxP (orange triangle) and lox2272 (green triangle) sites permit Cre-mediated recombination and inversion of the flanked transgene with respect to the EF-1α promoter. Downstream sequences stabilize the mRNA (woodchuck polyresponse element, WPRE) and trigger polyadenylation (human growth hormone polyadenylation, hGH polyA). After recombination, the transgene is flanked by one loxP and one lox2272 site, which do not recombine efficiently, effectively locking the transgene into position. The starting orientation of the transgene determines the Cre dependence of expression. The double-floxed orientation (1, DO) configuration, in which the open reading frame (ORF) of the transgene begins in the functional orientation with respect to the promoter, maintains expression only in cells lacking Cre (Cre-Off). In the opposite starting orientation, the double-floxed inverted (2, DIO) ORF must be recombined to be functional and expression is achieved only in Cre expressing cells (Cre-On). A single transgene containing two ORFs oriented oppositely with respect to each other and separated by stop codons (3, Cre-Switch) switches expression between the two ORFs depending on Cre expression. For Cre-Switch transgenes, the first, forward orientated ORF is expressed in Cre negative cells whereas the second, inverted ORF is activated in Cre positive cells. ITR = inverted terminal repeats (cyan). B. Cre-Off control of transgene expression can also be achieved by Cre-based excision of the ORF using alternative loxFAS sites. loxFAS sites (purple) flank the ORF and are oriented in the same direction such that the flanked sequence is excised by Cre. Figure and legend adapted from Saunders A, Johnson CA and Sabatini BL (2012) Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in neurons. Front. Neural Circuits 6:47. doi: 10.3389/fncir.2012.00047.
Figure 3
Figure 3. FAS rAAVs achieve Cre-Off expression compatible with Cre-On rAAVs or Cre-activated genomic alleles
A-B. Comparison of DO and FAS Cre-Off rAAV expression in conjunction with DIO Cre-On rAAVs and Cre-reporter alleles. Left, image of the primary infection. Right, normalized mean fluorescent values A. Simultaneous injection of DO/DIO vs. FAS/DIO rAAVs to test for differential Cre-Off/On transgene expression. rAAVs were mixed and injected into the striatum of D2r-Cre mouse, where Cre+ iSPNs are intermingled with Cre- dSPNs and interneurons. DIO-GFP and DO-mCherry co-injection (top) results in expression patterns which are segregated and antagonistic. DIO-GFP and FAS-tdTomato co-injection (bottom) results in expression patterns which are spatially congruent. B. Comparison of DO vs. FAS Cre-Off rAAV expression in a Cre-reporter mouse. DO-GFP and FAS-GFP rAAVs were independently injected into the cortex of PV i-Cre;Rosa26tdTomato(Ai19) mice. Cre expression in PV+ interneurons activates the tdTomato reporter through excision of loxP flanked stop cassette. In the area containing the DO-GFP infection, tdTomato reporter expression is greatly diminished. This inhibition is specific to DO rAAVs, as FAS-GFP infection does not affect reporter fluorescence. Figure adapted from Saunders A, Johnson CA and Sabatini BL (2012) Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in neurons. Front. Neural Circuits 6:47. doi: 10.3389/fncir.2012.00047.
Figure 4
Figure 4. Strategies for validating Cre-Off rAAVs
A-C. Cre-Off rAAVs are transcriptionally active until Cre mediated recombination inactivates transgene expression. Since Cre-Off efficiency is dependent on Cre concentrations in the nucleus, Cre-Off rAAVs should be validated for each driver line, brain region and developmental period for proper experimental interpretation. We suggest three methods of validation, anatomical (A), Immunohistochemical (IHC, B) and rAAV Cre-switch (C). We summarize these methods with cartoons in which each depicts fully efficient Cre-Off inactivation. The dotted boxes highlight the regions for image analysis. A. When the Cre+ neuron population has well described axonal or dendritic anatomy, and the full penetrance of Cre expression in that cell type has been confirmed, the experimenter can test Cre-Off expression through anatomical exclusion. For example, dopamine 1 receptor (D1r) expressing direct pathway spiny projection neurons (dSPNs) contribute the entire axonal projection from the striatum to the Substantia Nigra reticulata (SNr). To determine whether Cre targeted to dSPNs in D1r-Cre transgenic is a sufficient to inactivate rAAV FAS-tdTomato expression, one could assess the density of tdTomato+ axons in the SNr (dotted box) in comparison to a similar injection in a wild-type (WT) control mouse. Efficient inactivation would lead to an absence of tdTomato+ axons in the SNr, but should not affect the Cre- indirect pathway spiny projection neuron (iSPN) projection to the globus pallidus externus (GP). B. Many Cre driver lines reproduce endogenous patterns of the driver gene. If antibodies against the driver gene-products are available, IHC may be used to quantify exclusion of Cre-Off rAAV expression from IHC immunopositive marker+ cells. Alternatively, IHC can be performed against Cre itself. For example, to determine if FAS-tdTomato expression is inactivated from PV i-Cre interneurons of the cortex, following infection, IHC can be performed against Parvalbumin or Cre and degree of co-localization measured with confocal microscopy. Similar experiments in WT controls can inform to what degree the lack of co-localization is due to transduction inefficiency rather than inactivation. Importantly, transient Cre expression could inactivate rAAV expression but become undetectable at the time of IHC. C. Even with no anatomical knowledge or marker antibodies for a Cre+ population, Cre-switch rAAVs provide a well-controlled system for assaying recombination efficiency. For example, if interested in the cell population expressing Gene X in Brain Region Y, one could simply inject the Cre-Switch rAAV DIO-GFP_FAS-tdTomato and quantify the degree of co-localized GFP and tdTomato expression. Since transgene+ cells are typically transduced by more than one rAAV particle, low levels of Cre will recombine some but not all of the Cre-Switch genomes, leading to cells double positive for GFP and TdTomato. High levels of Cre will efficiently recombine all Cre-Switch genomes, leading to non-overlapping GFP+ and tdTomato+ cells.
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Internet Resources

    1. http://www.addgene.org/Bernardo_Sabatini/DO, FAS and Cre-Switch rAAVs available from AddGene

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