Figure 1.

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.