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. Author manuscript; available in PMC: 2014 Oct 15.
Published in final edited form as: Behav Brain Res. 2013 Mar 6;255:3–18. doi: 10.1016/j.bbr.2013.02.037

Figure 1. Diverse strategies for developing transgenic lines with functional expression of engineered opsins in the mammalian nervous system.

Figure 1

(A–C) Plasmid transgenic strategy. (A) An engineered opsin gene fused to a reporter gene (XFP) is inserted into a plasmid just downstream of the defined promoter region and upstream of a polyadenylation (pA) transcription termination sequence. (B) The Thy1.2 vector directs neuron-specific transgene expression. The opsin-XFP transgene is inserted downstream of the 4.2 Kb Thy1.2 promoter region. Deletion of exon III and flanking introns prevents expression in non-neuronal tissues. (C) Sagittal brain sections showing the broad transgene expression pattern for Th1.2-ChR2-EYFP line 18 (top) and Thy1.2-eNpHR2.0-EYFP line 2 (bottom). (D–F) BAC transgenic strategy. (D) Selection of a suitable 200 Kb clone from a BAC library which contains ~100 Kb of upstream sequence and ~50 Kb of downstream sequence flanking the ‘Target Gene.’ Note the presence of loxP and mlox511 sites in the BAC vector sequence, as well as hypothetical extra genes (Gene X, Gene Y). (E) BAC recombineering steps in the E. coli host cell, including homologous recombination using two short homology arms (Box A and Box B) to target the transgene cassette into the BAC DNA at a defined site, and Flp –mediated excision for removal of the FRT flanked selection cassette (NEO). (F) Sagittal brain sections from BAC transgenic mouse lines showing restricted cell-type specific transgene expression in cholinergic (left), serotonergic (middle), and Pvalb-expressing (right) neuronal subsets. (G) Knock-in strategy. Gene targeting involves homologous recombination in mouse ES cells. The transgene cassette is targeted to a specific endogenous chromosomal locus using a short homology arm (SHA) and long homology arm (LHA). The diphtheria toxin-A (DTA) gene is for negative selection against random integration events and the loxP flanked NEO cassette is for positive selection of correctly targeted clones. The NEO cassette is excised by expression of Cre.

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