Knock-in rats expressing Cre and Flp recombinases at the Parvalbumin locus

Abstract

Rats have the ability to learn and perform sophisticated behavioral tasks, making them very useful for investigating neural circuit functions. In contrast to the extensive mouse genetic toolkit, the paucity of recombinase-expressing rat models has limited the ability to monitor and manipulate molecularly-defined neural populations in this species. Here we report the generation and validation of two knock-in rat strains expressing either Cre or Flp recombinase under the control of Parvalbumin (Pvalb), a gene expressed in the critical "fast-spiking" subset of inhibitory interneurons (FSIs). These strains were generated with CRISPR-Cas9 gene editing and show highly specific and penetrant labeling of Pvalb-expressing neurons, as demonstrated by in situ hybridization and immunohistochemistry. We validated these models in both prefrontal cortex and striatum using both ex vivo and in vivo approaches, including whole-cell recording, optogenetics, extracellular physiology and photometry. Our results demonstrate the utility of these new transgenic models for a wide range of neuroscience experiments. IntroductionThe extensive library of recombinase driver mouse models [1,2] has enabled precise functional and structural dissection of neural circuits by providing specific and reliable genetic access to molecularly defined neural populations. When used in combination with recombinase-dependent reporter models or viruses, reporter genes can be expressed in specific neural populations [3]. For complex behavioral and large-scale in vivo electrophysiological experiments, rats are the model of choice. Yet their usefulness is restricted by the limited number of transgenic recombinase driver models to label molecularly defined neural populations. Thus, reliable rat recombinase driver models have great potential for accelerating progress in neuroscience.One important application of recombinase driver models is to suppress local brain activity in real time. This can be effectively achieved by optogenetic stimulation of local inhibitory interneurons, as demonstrated in mice using inhibitory neuron-specific Cre models [2,4]. In both cortical [5] and subcortical [6] regions, Parvalbumin (Pvalb) is expressed by GABAergic interneurons that provide perisomatic inhibition to projection neurons [7]. A rat recombinase driver model that directs transgene expression to Pvalb+ interneurons will therefore be a powerful tool for temporally-precise control of local circuit function.Until recently, it has been difficult to insert exogenous genes of interest into specific genomic locations in rats, primarily due to the lack of pluripotent stem cells for in vitro genomic modification [8]. To overcome this limitation, genetically modified rats have been predominantly generated using Bacterial Artificial Chromosome (BAC) transgenic technology [9][10][11]. This involves inserting a large chunk of chromosomal DNA (200-300kb) into the genome that includes the promoter of interest, coupled with an additional coding sequence for the desired recombinase. Expres...

Optogenetic pacing of medial septum parvalbumin-positive cells disrupts temporal but not spatial firing in grid cells

“…To target the GABAergic projection from MSA to MEC, channelrhodopsin (ChR2) was selectively expressed in PV + cells in MSA by injecting a viral construct in four animals from a Pvalb Cre rat line (Fig. 1A) (32). Immunohistochemical labeling of ChR2 confirmed that virus expression was largely restricted to MSA (Fig.…”

“…We used male Long-Evans Pvalb Cre knock-in rats (3 to 8 months old, 350 to 550 g at surgery) (32). An internal ribosomal entry site-Cre recombinase sequence cassette was inserted into the Pvalb locus using CRISPR-Cas9 gene editing.…”

“…We hypothesized that by controlling the spiking activity of grid cells through disinhibition, thus locking the spike-phase relation to the stimulation frequency, phase precession would also be abolished. We restricted the perturbation to the GABAergic part of the MSA projections by taking advantage of the Cre-Lox system using a Pvalb Cre rat line (32). This allowed us to selectively target inhibitory cell types expressing parvalbumin (PV), the main GABAergic cell type in MSA (33,34).…”

Beyond knockouts: Applying precision genome editing for conditional mutagenesis innovation in zebrafish

“…Our results showed that the frequency of germline transmission of precise integration events for ascl1b, olig2 and neurod1 were 33%, 5% and 20%, respectively, without the need to screen more than 20 adult fish for any of the three lines. This is in contrast to previous studies reporting lower efficiencies using similar CRISPR-targeting strategies to isolate Cre transgenics (Hasegawa et al, 2016;Yu et al, 2018;Hu et al, 2019;Kaiser and Feng, 2019;Pettibone et al, 2019). The variable efficiencies may be due to different methods of Cas9 and sgRNA delivery (RNA or plasmid), and donor repair template design (linear, circular, ssDNA, dsDNA, with or without sgRNA cut sites).…”

“…Particularly important, these previously reported methods did not show a consensus 114 regarding homology length. Yu et al (2018) developed two CRISPR-knock in lines in rat, Pvalb:Cre and Pvalb:Flp, using dsDNA plasmids with different homology lengths, 1167 bp/1693 bp and 794 bp/469 bp, respectively. Kaiser and Feng (2019) also developed two different CRISPR-knock in lines in mice for the same gene.…”