The main limitation of this approach is that transgene

expression often does not fully recapitulate that of the endogenous gene and varies among transgenic lines. Given current knowledge about mammalian gene regulation and chromatin biology, this should perhaps come as no surprise. First, cis regulatory elements (enhancers, repressors, insulators) are often very distant from the transcription start site MEK inhibitor ( Kapranov et al., 2007); thus, even BAC constructs often do not contain the full complements of regulatory elements of the gene of interest. Second, because cis-regulatory elements can act at a very long distance ( Bulger and Groudine, 2011 and Heintzman and Ren, 2009), enhancers and repressors near the transgene integration site (but that are unrelated to the promoter elements in the transgene) can influence transcription, leading to ectopic or suppressed expression. Third, different transgenic lines will have different expression patterns due to differential enhancer/repressor influences at different genomic integration sites. Fourth, transgenes inserted into a foreign chromatin environment can be silenced or epigenetically altered in unpredictable ways. Thus, the challenges often associated with the transgenic strategy are the uncertainty of the targeted

neurons and the effort necessary to ascertain their identity and property. Here, we have used the gene knockin strategy to target GABAergic neurons. Cre cassettes are inserted by homologous recombination at endogenous gene loci, which are embedded in their native chromatin ABT-263 research buy environment with largely intact regulatory elements. The main advantage of this strategy is

that Cre expression precisely and reliably recapitulates the targeted endogenous gene. Indeed, after extensive characterization we found that recombination patterns in almost all the Cell press GABA Cre drivers often perfectly match the spatial and temporal pattern of the endogenous gene expression. The disadvantages of gene targeting approach include: (1) the possibility of altering the expression of the targeted gene, even when a bicistronic cassette (e.g., ires or T2A) is inserted after the targeted gene (see below); (2) the full expression pattern of a gene may include multiple cell types or brain regions; thus, in some cases the partial expression pattern may be more desirable (although this issue can sometimes be addressed by using Cre-dependent viral vectors which can be injected to defined brain regions). Extensive characterization of eight constitutive drivers indicated that this strategy is highly effective. First, Cre activities appear highly specific and largely match the expression of the targeted genes. In certain lines and brain regions, recombination patterns do deviate from that of the endogenous expression in adult brain (e.g., CCK-ires-Cre, Figure 6D).