PMC

Co-infection with DIO or DO/FAS rAAVs drives co-expression without spatial interference. (A) Infection of multiple DIO Cre-On rAAVs results in co-expression of transgenes without interference. Left, a sagittal section of a D2-Cre mouse infected with DIO-GFP and DIO-mCherry in the striatum. Right, normalized mean fluorescent values for GFP and mCherry quantified from boxed inset in A. (B–D) Infection of multiple Cre-Off rAAVs results in co-expression of transgenes without spatial interference. Left, sagittal sections from wild type mice infected with B. DO-mCherry + DO-GFP, C. DO-mCherry + FAS-GFP, or D. FAS-TdTomato + FAS-GFP in the striatum. Right, normalized mean fluorescent values for GFP and mCherry/TdTomato quantified from boxed inset in to the left.

A single orientation switching transgene can mediate Cre-On and Cre-Off expression without spatial interference. (A) Left, Sagittal section through a D1-Cre mouse infected with a Cre-Switch transgene encoding Cre-Off TdTomato and Cre-On GFP in the striatum. Right, more medial section containing the GP and both TdTomato⁺ and GFP⁺ projections. Bottom, GFP and TdTomato fluorescence is spatially intermingled. (B) Normalized mean fluorescent values for GFP and TdTomato quantified from boxed inset in A, bottom right. (C) Confocal imaging through the infection volume (N = 3 infections, N = 796 cells) revealed that GFP (N = 489 cells) and TdTomato (N = 302 cells) were expressed in largely non-overlapping cell populations (N = 7/791 cells were double-positive for GFP and TdTomato).

Cre-Off infection with DO rAAV interferes with the expression of fluorescent Cre reporter allele whereas infection with FAS rAAV does not. (A) Coronal section through the cortex of PV-Cre; Ai9-lsl-TdTomato mouse after infection with DO-GFP. Separate GFP and TdTomato fluorescent channels demonstrate that Cre reporter fluorescence is drastically reduced in the area of DO-GFP infection. (B) Normalized mean fluorescent values for GFP and TdTomato quantified from boxed inset in A. (C) Coronal section through the cortex of PV-Cre; Ai9-lsl-TdTomato mouse after infection with FAS-GFP. Separate GFP and TdTomato fluorescent channels demonstrate that Cre reporter fluorescence is not reduced in the area of FAS-GFP infection. (D) Normalized mean fluorescent values for GFP and TdTomato quantified from boxed inset in C. (E) DO-GFP expression is excluded from remaining TdTomato⁺ cells in the conditions shown in (A). Top, a single confocal plane at the border region of the infection containing both TdTomato⁺ and GFP⁺ cells. Bottom, confocal imaging of volumes of infected tissue (N = 2 infections, N = 527 GFP⁺ cells, N = 43 TdTomato⁺ cells) reveal GFP expression is excluded from TdTomato⁺ cells. (F) FAS-GFP expression is excluded from TdTomato⁺ cells in the conditions shown in C. Top, a single confocal plane of the infection containing both TdTomato⁺ and GFP⁺ cells. Bottom, confocal imaging of volumes of infected tissue (N = 2 infections, N = 177 GFP⁺ cells, N = 105 TdTomato⁺ cells) reveal GFP expression is almost completely excluded from TdTomato⁺ cells (N = 1/282 cells double-positive for GFP and TdTomato).

Strategies to achieve Cre-dependent rAAV transgene expression. (A) Oppositely oriented loxP (gray triangle) and lox2272 (black 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. (B) Cre-Off control of transgene expression can also be achieved by Cre-based excision of the ORF using alternative lox FAS sites. loxFAS sites flank the ORF and are oriented in the same direction such that the flanked sequence is excised by Cre.

LoxFAS excision achieves efficient Cre-Off rAAV expression that does not interfere with DIO Cre-On expression. (A) Sagittal section through a D2-Cre mouse infected with FAS-TdTomato in the striatum. TdTomato is excluded from iMSNs. TdTomato⁺ axons arborize in the SNr (inset) but only minimally in the GP, consistent with expression in dMSNs. (B) Sagittal section through a D1-Cre mouse infected with FAS-TdTomato in the striatum. TdTomato expression is excluded from dMSNs. TdTomato⁺ axons leaving the striatum target the GP, but not the SNr (inset), consistent with exclusion from dMSNs. (C) Antibody staining against Cre in D2-Cre striatum infected with FAS-TdTomato reveals efficient Cre-Off expression in the presence of Cre. Top, a single confocal plane showing Cre⁺ nuclei excluded from interspersed TdTomato⁺ cells. Bottom, confocal imaging through infected and stained tissue volumes (N = 4 infections, N = 929 cells) revealed that TdTomato expression (N = 477 cells) was completely absent in Cre⁺ cells (N = 452). (D) Co-infection of rAAVs Cre-GFP and FAS-TdTomato into the striatum of a wild type (WT) mouse leads to efficient exclusion of TdTomato in Cre-GFP expressing cells. Top, a single confocal plane containing interspersed GFP⁺ and TdTomato⁺ cells. Note that the GFP expression is nuclear due to the targeting of Cre. The arrowheads indicate cells co-expressing GFP and TdTomato. Bottom, confocal imaging of volumes of infected tissue (four infections, N = 1258 cells) reveal that a small fraction of the total GFP expressing cells (N = 948) also expressed TdTomato (N = 40 cells). Conversely, the majority of TdTomato⁺ cells (N = 610) did not express Cre-GFP. (E) Sagittal section through a D2-Cre mouse infected with DIO-GFP and FAS-TdTomato in the striatum. GFP⁺ axons arborize in the GP, whereas TdTomato⁺ axons pass through GP en route to the SNr. Bottom, GFP, and TdTomato signals exhibit spatially intermingled expression patterns. (F) Normalized mean fluorescent values for GFP and TdTomato quantified from boxed inset in E, bottom right. GFP and TdTomato expression levels mirror each other throughout the infection area. (G) Confocal imaging through infected volumes (N = 3 infections, N = 1116 cells) revealed that DIO and FAS co-infection targeted expression to mostly non-overlapping cell populations (FAS-TdTomato, N = 578 cells; DIO-GFP, N = 519; FAS-TdTomato and DIO-GFP, N = 19).

DIO and DO rAAVs achieve Cre-On and Cre-Off expression but exhibit interfering interactions when co-infected. (A) Sagittal section through a D2-Cre mouse infected with DIO-GFP in the striatum. GFP⁺ axons emanating from the striatal infection innervate the globus pallidus (GP), the target of indirect pathway medium spiny neurons (iMSNs). No GFP⁺ expression is observed in the substantia nigra reticulata (SNr, inset). COR = cortex, STR = striatum. (B) Sagittal section through a D2-Cre mouse infected with DO-mCherry in the striatum such that mCherry expression is excluded from iMSNs. mCherry⁺ axons leaving the striatum innervate the SNr (inset), consistent with expression in direct pathway MSNs (dMSNs). (C) Antibody staining against Cre in D2-Cre striatum infected with DO-mCherry reveals that Cre efficiently turns off mCherry expression, indicative of Cre-Off behavior. Top, a single confocal plane showing mCherry expression excluded from cells with Cre⁺ nuclei. Bottom, quantification of co-expression of Cre and mCherry (N = 4 infections, N = 1187 cells) reveals that mCherry expression (N = 495 cells) was almost completely excluded from Cre⁺ cells (N = 1/691 cells double-positive for mCherry and Cre). (D) Co-infection of rAAVs encoding a Cre-mCherry fusion and DO-GFP into the striatum of a wild type (WT) mouse efficiently prevents GFP expression in Cre-mCherry expressing cells. Top, a single confocal plane containing interspersed mCherry⁺ and GFP⁺ cells. Note that the mCherry expression is nuclear due to nuclear targeting of Cre. The arrowhead indicates a cell co-expressing mCherry and GFP. Bottom, confocal imaging of infected tissue (N = 2 infections, N = 521 cells) reveals that cells expressing Cre-mCherry (N = 237) and cells expressing GFP (N = 284) are largely non-overlapping (N = 11/521 cells were double-positive for both fluorophores). (E) Sagittal section through a D2-Cre mouse infected with DIO-mCherry and DO-GFP in the striatum. Despite the physical intermixing of Cre positive and negative neurons in the striatum, the resulting GFP and mCherry expression is spatially segregated. Below, separate visualization of the green and red fluorescence channels demonstrates that mCherry is excluded from the striatal volume expressing GFP. (F) Normalized mean fluorescent values for GFP and mCherry quantified from boxed inset in E, bottom right. (G) Confocal imaging around the border regions (N = 2 infections, N = 865 cells) containing both GFP⁺ (N = 529) and mCherry⁺ (N = 336) cells revealed no overlap in expression.