Figure 2. Transgene-independent strategies for enhanced opsin expression in transgenic animals.

(A) Combination of the CAG promoter and WPRE sequence enhances transgene expression from Cre-inducible Rosa26 knock-in alleles. The presence of a floxed STOP cassette prevents opsin-XFP expression in the absence of Cre. Following breeding of the Cre reporter and Cre driver lines, the floxed STOP is excised in Cre-expressing cells to allow for opsin-XFP expression. Please see ref. 74 and ref. 75 for full details. (B) the KENGE-tet improved tTA-inducible transgene expression system utilizes the gene targeting method to insert a tetO-opsin-XFP-pA cassette immediately downstream of the beta-actin (Actb) pA sequence. Following breeding of the tet responsive line with the a cell-type specific tTA driver line, opsin-XFP expression is activated in tTA-expressing cells (ref. 82). (C) Controlling gene dosage with viral 2A peptide linkers. One (1X), two (2X), or three (3X) copies of the opsin gene are linked to an upstream cellular promoter region and downstream reporter gene and polyadenylation (pA) sequence. The 2A peptide linkers ensure separate stoichiometric expression of the opsin and XFP protein. This strategy was modified from ref. 76.