Transgenic Driver Research Articles

A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types.

The mammalian cortex is comprised of cells classified into types according to shared properties. Defining the contribution of each cell type to the processes guided by the cortex is essential for understanding its function in health and disease. We used transcriptomic and epigenomic cortical cell type taxonomies from mouse and human to define marker genes and putative enhancers and created a large toolkit of transgenic lines and enhancer AAVs for selective targeting of cortical cell populations. We report evaluation of fifteen new transgenic driver lines, two new reporter lines, and >800 different enhancer AAVs covering most subclasses of cortical cells. The tools reported here as well as the scaled process of tool creation and modification enable diverse experimental strategies towards understanding mammalian cortex and brain function.

A safety guide for transgenic Cre drivers in metabolism.

A safety guide for transgenic Cre drivers in metabolism.

Type selective ablation of postnatal slow and fast fatigue-resistant motor neurons in mice induces late onset kinetic and postural tremor following fiber-type transition and myopathy

Animals on Earth need to hold postures and execute a series of movements under gravity and atmospheric pressure. VAChT-Cre is a transgenic Cre driver mouse line that expresses Cre recombinase selectively in motor neurons of S-type (slow-twitch fatigue-resistant) and FR-type (fast-twitch fatigue-resistant). Sequential motor unit recruitment is a fundamental principle for fine and smooth locomotion; smaller-diameter motor neurons (S-type, FR-type) first contract low-intensity oxidative type I and type IIa muscle fibers, and thereafter larger-diameter motor neurons (FInt-type, FF-type) are recruited to contract high-intensity glycolytic type IIx and type IIb muscle fibers. To selectively eliminate S- and FR-type motor neurons, VAChT-Cre mice were crossbred with NSE-DTA mice in which the cytotoxic diphtheria toxin A fragment (DTA) was expressed in Cre-expressing neurons. The VAChT-Cre;NSE-DTA mice were born normally but progressively manifested various characteristics, including body weight loss, kyphosis, kinetic and postural tremor, and muscular atrophy. The progressive kinetic and postural tremor was remarkable from around 20 weeks of age and aggravated. Muscular atrophy was apparent in slow muscles, but not in fast muscles. The increase in motor unit number estimation was detected by electromyography, reflecting compensatory re-innervation by remaining FInt- and FF-type motor neurons to the orphaned slow muscle fibers. The muscle fibers gradually manifested fast/slow hybrid phenotypes, and the remaining FInt-and FF-type motor neurons gradually disappeared. These results suggest selective ablation of S- and FR-type motor neurons induces progressive muscle fiber-type transition, exhaustion of remaining FInt- and FF-type motor neurons, and late-onset kinetic and postural tremor in mice.

18. Tissue Microenvironment Affects Fibrotic Capsules Formed during Foreign Body Response

PURPOSE: Foreign body response (FBR) is a common surgical complication, wherein a fibrotic capsule forms around an implant. Though it is posited that FBR has mechanistic overlap with normal wound healing, the specific cell populations responsible for this phenomenon remain elusive. Our group previously demonstrated that En1 positive fibroblasts (EPFs) predominate in murine dorsal wounds, and showed that these cells are responsible for scar formation within the wound bed. This observation led us to explore the role of EPFs and other fibroblast subpopulations during FBR. METHODS: En-1Cre transgenic driver mice were crossed with R26mTmG reporter mice, generating En-1Cre; Rosa26mTmG (En1+) mice. In this mouse line, cells expressing En1 also express GFP, while all others express RFP. Silicone discs were surgically implanted below the subcutaneous layer of En1+ mice dorsi, and fibrotic capsules surrounding the implants were harvested at POD28. Immunohistochemistry and flow cytometry (FACS) analysis were utilized to evaluate the cellular characteristics of the En1+ cells. A subset of fibrotic capsules placed in C57BL/6J for 28 days was also prepared for single-cell RNA sequencing. Software packages in R and Python were used to identify and define subtypes of fibroblasts present within fibrotic capsules. Crosstalk and signaling pathways were further identified via network analysis platforms. RESULTS: Immunohistochemistry and FACS analysis confirmed that En1+ cells predominate in the fibrotic capsule, and that GFP+ cells also expressed pro-fibrotic markers, including Col-1 and α-SMA. As these cells were also negative for the endothelial and immune cell markers CD41 and CD45, respectively, these data indicate that En+ cells within capsules were primarily of fibroblast origin. Interestingly, basic histology highlighted that capsule thickness (p < 0.0001; n = 4) and collagen deposition (p = 0.0016; n = 4) were higher in the capsule adjacent to the subcutaneous tissue (subcutaneous capsule) in comparison to that adjacent to muscle tissue (muscle capsule). In parallel, our transcriptional dataset revealed that the distribution of fibroblast subpopulations present in subcutaneous capsule to that in muscle capsule differed. Notably, cell clustering, cell-cell interaction, and gene ontology analyses underscored that fibroblasts in the subcutaneous capsules were more fibrotic in nature relative to those present in muscle capsule. CONCLUSION: These data strongly suggest that, as with normal wound healing in which EPFs are diffusely present within a healing scar, En1 appears to play a key role in fibrotic capsule formation. Further, the presence of multiple subpopulations of fibroblasts revealed by our transcriptional analysis underscores the significant function fibroblasts may have in FBR, and that the tissue microenvironment may play a role in the fibrotic response. Deeper understanding of the mechanism behind fibroblasts’ involvement and the effect the tissue microenvironment may have on fibrotic capsule formation may offer potential therapeutic targets to reduce FBR post surgical implantation in the future.

A sex difference in mouse dopaminergic projections from the midbrain to basolateral amygdala

BackgroundDopaminergic circuits play important roles in the motivational control of behavior and dysfunction in dopaminergic circuits have been implicated in several psychiatric disorders, such as schizophrenia and depression. While these disorders exhibit different incidence rates in men and women, the potential sex differences in the underlying neural circuits are not well-understood. Previous anatomical tracing studies in mammalian species have revealed a prominent circuit projection connecting the dopaminergic midbrain ventral tegmental area (VTA) to the basolateral amygdala (BLA), which is involved in emotional processing and associative learning. However, whether there is any sex difference in this anatomical circuit remains unknown.MethodsTo study the potential sex differences in the VTA-to-BLA dopaminergic circuit, we injected two viral vectors encoding fluorescent reporters of axons and synaptic boutons (AAV–FLEX–tdTomato and AAV–FLEX–SynaptophysinGFP, respectively) into the VTA of a mouse transgenic driver line (tyrosine hydroxylase promoter-driven Cre, or TH-Cre), which restricts the reporter expression to dopaminergic neurons. We then used confocal fluorescent microscopy to image the distribution and density of dopaminergic axons and synaptic boutons in serial sections of both male and female mouse brain.ResultsWe found that the overall labeling intensity of VTA-to-BLA dopaminergic projections is intermediate among forebrain dopaminergic pathways, significantly higher than the projections to the prefrontal cortex, but lower than the projections to the nucleus accumbens. Within the amygdala areas, dopaminergic axons are concentrated in BLA. Although the size of BLA and the density of dopaminergic axons within BLA are similar between male and female mice, the density of dopaminergic synaptic boutons in BLA is significantly higher in male brain than female brain.ConclusionsOur results demonstrate an anatomical sex difference in mouse dopaminergic innervations from the VTA to BLA. This finding may provide a structural foundation to study neural circuit mechanisms underlying sex differences in motivational and emotional behaviors and related psychiatric dysfunctions.

Adipocytes the Forgotten Culprit in Skin Fibrosis: Exploring the Mechanism of Fat Driven Skin Fibrosis

INTRODUCTION: Adipocytes have previously been implicated in skin wound healing in both metabolic and nonmetabolic roles. We hypothesized that mature adipocytes directly participate in wound repair via conversion into fibroblasts and that adipocyte-derived fibroblasts contribute to skin scarring. METHODS: AdipoqCre transgenic driver mice were crossed to R26mTmG reporter mice to generate AdipoqCreERT;ROSA26mTmG mice to perform lineage tracing of mature adipocytes. To achieve local adipocyte ablation AdipoqCre;ROSA26mTmG;R26tm1 (HBEGF) Awai mice were generated and wounded, and diphtheria toxin (DT) was injected into the wound base. DT- and vehicle control-treated wounds underwent histologic analysis. Last, we performed scRNAseq on wounded and unwounded tissue to identify fibrotic fibroblast subpopulations. RESULTS: Using our AdipoqCre;ROSA26mTmG adipocyte lineage-tracing model (Fig. A), we identified significantly greater number of adiponectin lineage-positive cells (GFP+) within wounds at postoperative day 14 (POD-14) compared with unwounded skin (*p < 0.05, n = 6; Fig. B). FACS further confirmed that the GFP+ cells were fibroblasts and increased to 10% at POD-14 (Fig. C). Compared with typical subcutaneous adipocytes, the GFP+ cells exhibited upregulation of fibroblast markers and downregulation of adipocyte markers (Fig. D). DT-induced ablation of Adipoq lineage-positive cells (Fig. E) exhibited reduced scar thickness (Fig. F) and collagen deposition at POD-14 compared with control wounds (*p < 0.05, n = 6; Fig. G). scRNAseq analysis revealed novel markers for distinct mechanosensitive fibroblast subpopulations responsible for scar formation (Fig. H).FigureCONCLUSION: Our findings strongly suggest that mature adipocytes in the skin undergo conversion to profibrotic fibroblasts in response to injury. Local adipocyte ablation resulted in reduced scarring, suggesting that adipocyte-derived fibroblasts are important contributors to wound fibrosis. These findings together suggest that adipocyte mechanosensitive fibroblast subpopulations drive dermal scar formation.

Engrailed-Positive Fibroblasts: The Primary Cell Type Present in Fibrotic Capsules During Foreign Body Response

Introduction: We have recently shown that En1 positive fibroblasts (EPFs) predominate in wounds and are responsible for scar formation. Meanwhile, foreign body response (FBR) is a common surgical complication whereby a fibrotic capsule forms around an implant. Though believed to have some mechanistic overlap with normal wound healing, the specific cell populations responsible are unknown. Methods: En1Cre transgenic driver mice were crossed with R26mTmG reporter mice, generating En1Cre; Rosa26mTmG (En1+) mice. In these mice, cells that express En1 also express GFP, while all other cells express RFP. Silicone discs were surgically implanted below the subcutaneous layer of En1+ mice dorsi, and fibrotic capsules surrounding the implants were harvested at POD30. Immunohistochemistry and flow cytometry (FACS) analysis were used to evaluate the cellular characteristics of the En1+ cells. Results: Using immunohistochemistry, En1+ cells were found to be more prevalent in the fibrotic capsule relative to unwounded tissue (*P < 0.05, n = 6). Furthermore, immunofluorescence staining and FACS revealed that GFP+ cells within the fibrotic capsules expressed pro-fibrotic markers such as Col-I, a-SMA, and Col-III. These cells were also negative for CD41 and CD45, endothelial and immune cell markers, respectively. These data indicate that En1+ cells seen within fibrotic capsules were primarily of fibroblast origin. Conclusion: As with normal wound healing, where EPFs are prevalent within a healing scar, our findings strongly suggest that En1 plays a key role in fibrotic capsule formation. Further understanding of the mechanisms that drive En1 positive fibroblasts towards fibrosis may offer potential therapeutic targets to prevent fibrotic capsule formation.

LATS1/2 control TGFB-directed epithelial-to-mesenchymal transition in the murine dorsal cranial neuroepithelium through YAP regulation.

Hippo signaling, an evolutionarily conserved kinase cascade involved in organ size control, plays key roles in various tissue developmental processes, but its role in craniofacial development remains poorly understood. Using the transgenic Wnt1-Cre2 driver, we inactivated the Hippo signaling components Lats1 and Lats2 in the cranial neuroepithelium of mouse embryos and found that the double conditional knockout (DCKO) of Lats1/2 resulted in neural tube and craniofacial defects. Lats1/2 DCKO mutant embryos had microcephaly with delayed and defective neural tube closure. Furthermore, neuroepithelial cell shape and architecture were disrupted within the cranial neural tube in Lats1/2 DCKO mutants. RNA sequencing of embryonic neural tubes revealed increased TGFB signaling in Lats1/2 DCKO mutants. Moreover, markers of epithelial-to-mesenchymal transition (EMT) were upregulated in the cranial neural tube. Inactivation of Hippo signaling downstream effectors, Yap and Taz, suppressed neuroepithelial defects, aberrant EMT and TGFB upregulation in Lats1/2 DCKO embryos, indicating that LATS1/2 function via YAP and TAZ. Our findings reveal important roles for Hippo signaling in modulating TGFB signaling during neural crest EMT.

68. Adipocytes Transition to Pro-fibrotic Fibroblasts and Contribute to Muscle Fibrosis Following Nerve Injury

Purpose: Recent studies have suggested that interconversion between adipocytes and fibroblasts (the cellular mediators of skin scarring) occurs in the wound environment. Muscle fibrosis occurs following aging, muscle injury, and muscular dystrophies causing weakness and pain. The role of adipocytes in muscle fibrosis has not been fully explored. Our study examines whether mature adipocytes participate in muscle fibrosis following nerve injury via conversion into dermal fibroblasts. Methods:AdipoqCre transgenic driver mice were crossed to R26mTmG reporter mice to generate AdipoqCre;ROSA26mTmG mice, in which mature adipocytes (Adipoq/adiponectin-expressing cells) express green fluorescent protein (GFP), and all other cells express a red fluorescent protein (RFP). AdipoqCre;ROSA26mTmG mice underwent sciatic nerve injury per established protocol. Injured tibialis anterior and gluteus maximus muscle was harvested at postoperative day-14 (POD14) and underwent immunofluorescent staining for fibroblast (collagen type IV/col-IV, alpha-smooth muscle actin/a-SMA) and adipocyte (perilipin) cell markers. To achieve local adipocyte ablation, AdipoqCre;ROSA26mTmG;R26tm1(HBEGF)Awai mice were generated and wounded, and diphtheria toxin (DT;400ng/mouse) was injected into the muscle at the nerve injury site. DT- and vehicle control-treated muscle underwent histologic analysis. Results: We identified a significantly greater number of adiponectin-lineage-positive cells (GFP+) within injured muscle at POD14 compared to uninjured muscle (*p<0.05,n=5). The GFP+ cells that infiltrated the muscle injury exhibited upregulation of fibrotic markers including col-IV and α-SMA, and downregulation of adipocyte markers including perilipin, indicating conversion of adipocytes into pro-fibrotic fibroblasts. DT-induced ablation of Adipoq-lineage-positive cells resulted in reduced muscle scarring, with decreased collagen deposition compared to vehicle-control injured muscle (*p<0.05,n=5). Conclusion: Our study demonstrates that adipocytes undergo conversion to pro-fibrotic fibroblasts in response to nerve injury and contribute to muscle fibrosis. Targeted adipocyte ablation resulted in reduced muscle scarring, suggesting that adipocyte-derived fibroblasts are important contributors to muscle fibrosis.

A non-disruptive and efficient knock-in approach allows fate tracing of resident osteoblast progenitors during repair of vertebral lesions in medaka.

During bone development and repair, osteoblasts are recruited to bone deposition sites. To identify the origin of recruited osteoblasts, cell lineage tracing using Cre/loxP recombination is commonly used. However, a confounding factor is the use of transgenic Cre drivers that do not accurately recapitulate endogenous gene expression or the use of knock-in Cre drivers that alter endogenous protein activity or levels. Here, we describe a CRISPR/Cas9 homology-directed repair knock-in approach that allows efficient generation of Cre drivers controlled by the endogenous gene promoter. In addition, a self-cleaving peptide preserves the reading frame of the endogenous protein. Using this approach, we generated col10a1p2a-CreERT2 knock-in medaka and show that tamoxifen-inducible CreERT2 efficiently recombined loxP sites in col10a1 cells. Similar knock-in efficiencies were obtained when two unrelated loci (osr1 and col2a1a) were targeted. Using live imaging, we traced the fate of col10a1 osteoblast progenitors during bone lesion repair in the medaka vertebral column. We show that col10a1 cells at neural arches represent a mobilizable cellular source for bone repair. Together, our study describes a previously unreported strategy for precise cell lineage tracing via efficient and non-disruptive knock-in of Cre.

Transgenic mice encoding modern imaging probes: Properties and applications.

SUMMARYModern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

Novel Mouse Models of Bladder Cancer Identify a Prognostic Signature Associated with Risk of Disease Progression.

To study the progression of bladder cancer from non-muscle-invasive to muscle-invasive disease, we have developed a novel toolkit that uses complementary approaches to achieve gene recombination in specific cell populations in the bladder urothelium in vivo, thereby allowing us to generate a new series of genetically engineered mouse models (GEMM) of bladder cancer. One method is based on the delivery of adenoviruses that express Cre recombinase in selected cell types in the urothelium, and a second uses transgenic drivers in which activation of inducible Cre alleles can be limited to the bladder urothelium by intravesicular delivery of tamoxifen. Using both approaches, targeted deletion of the Pten and p53 tumor suppressor genes specifically in basal urothelial cells gave rise to muscle-invasive bladder tumors. Furthermore, preinvasive lesions arising in basal cells displayed upregulation of molecular pathways related to bladder tumorigenesis, including proinflammatory pathways. Cross-species analyses comparing a mouse gene signature of early bladder cancer with a human signature of bladder cancer progression identified a conserved 28-gene signature of early bladder cancer that is associated with poor prognosis for human bladder cancer and that outperforms comparable gene signatures. These findings demonstrate the relevance of these GEMMs for studying the biology of human bladder cancer and introduce a prognostic gene signature that may help to stratify patients at risk for progression to potentially lethal muscle-invasive disease. SIGNIFICANCE: Analyses of bladder cancer progression in a new series of genetically engineered mouse models has identified a gene signature of poor prognosis in human bladder cancer.

A quick and versatile protocol for the 3D visualization of transgene expression across the whole body of larval Drosophila

Larval Drosophila are used as a genetically accessible study case in many areas of biological research. Here we report a fast, robust and user-friendly procedure for the whole-body multi-fluorescence imaging of Drosophila larvae; the protocol has been optimized specifically for larvae by systematically tackling the pitfalls associated with clearing this small but cuticularized organism. Tests on various fluorescent proteins reveal that the recently introduced monomeric infrared fluorescent protein (mIFP) is particularly suitable for our approach. This approach comprises an effective, low-cost clearing protocol with minimal handling time and reduced toxicity in the reagents employed. It combines a success rate high enough to allow for small-scale screening approaches and a resolution sufficient for cellular-level analyses with light sheet and confocal microscopy. Given that publications and database documentations typically specify expression patterns of transgenic driver lines only within a given organ system of interest, the present procedure should be versatile enough to extend such documentation systematically to the whole body. As examples, the expression patterns of transgenic driver lines covering the majority of neurons, or subsets of chemosensory, central brain or motor neurons, are documented in the context of whole larval body volumes (using nsyb-Gal4, IR76b-Gal4, APL-Gal4 and mushroom body Kenyon cells, or OK371-Gal4, respectively). Notably, the presented protocol allows for triple-color fluorescence imaging with near-infrared, red and yellow fluorescent proteins.

Molecular classification of zebrafish retinal ganglion cells links genes to cell types to behavior

Retinal ganglion cells (RGCs) form an array of feature detectors, which convey visual information to central brain regions. Characterizing RGC diversity is required to understand the logic of the underlying functional segregation. Using single-cell transcriptomics, we systematically classified RGCs in adult and larval zebrafish, thereby identifying marker genes for >30 mature types and several developmental intermediates. We used this dataset to engineer transgenic driver lines, enabling specific experimental access to a subset of RGC types. Expression of one or few transcription factors often predicts dendrite morphologies and axonal projections to specific tectal layers and extratectal targets. Invivo calcium imaging revealed that molecularly defined RGCs exhibit specific functional tuning. Finally, chemogenetic ablation of eomesa+ RGCs, which comprise melanopsin-expressing types with projections to a small subset of central targets, selectively impaired phototaxis. Together, our study establishes a framework for systematically studying the functional architecture of the visual system.

Regulatory Elements Inserted into AAVs Confer Preferential Activity in Cortical Interneurons.

Cortical interneuron (CIN) dysfunction is thought to play a major role in neuropsychiatric conditions like epilepsy, schizophrenia and autism. It is therefore essential to understand how the development, physiology, and functions of CINs influence cortical circuit activity and behavior in model organisms such as mice and primates. While transgenic driver lines are powerful tools for studying CINs in mice, this technology is limited in other species. An alternative approach is to use viral vectors such as AAV, which can be used in multiple species including primates and also have potential for therapeutic use in humans. Thus, we sought to discover gene regulatory enhancer elements (REs) that can be used in viral vectors to drive expression in specific cell types. The present study describes the systematic genome-wide identification of putative REs (pREs) that are preferentially active in immature CINs by histone modification chromatin immunoprecipitation and sequencing (ChIP-seq). We evaluated two novel pREs in AAV vectors, alongside the well-established Dlx I12b enhancer, and found that they drove CIN-specific reporter expression in adult mice. We also showed that the identified Arl4d pRE could drive sufficient expression of channelrhodopsin for optogenetic rescue of behavioral deficits in the Dlx5/6+/− mouse model of fast-spiking CIN dysfunction.

Unexpected effects of ivermectin and selamectin on inducible CreER activity in mice

BackgroundAnti-parasitics are frequently used in research animal facilities to treat a multitude of common infections, with pinworms and fur mites being amongst the most common. Ivermectin and selamectin are common oral and topical treatments for these infections, respectively. Although commonly thought to be innocuous to both the research animals and any transgenic elements that the animals may carry, evidence exists that ivermectin is capable of activating the recombinase activity of at least one CreER. The goal of the current study was to determine if there was an effect of either anti-parasitic agent on the activity of CreER proteins in transgenic mice.Case presentationWe analyzed the offspring of transgenic mice exposed to either ivermectin or selamectin during pregnancy and nursing. Through analysis of reporter genes co-expressed with multiple, independently generated transgenic CreER drivers, we report here that ivermectin and selamectin both alter recombinase activity and thus may have unintended consequences on gene inactivation studies in mice.ConclusionsAlthough the mechanisms by which ivermectin and selamectin affect CreER activity in the offspring of treated dams remain unclear, the implications are important nonetheless. Treatment of pregnant transgenic mice with these anti-parasitics has the potential to alter transgene activity in the offspring. Special considerations should be made when planning treatment of transgenic mice with either of these pharmacologics.

Newly Identified Electrically Coupled Neurons Support Development of the Drosophila Giant Fiber Model Circuit.

The Drosophila giant fiber (GF) escape circuit is an extensively studied model for neuron connectivity and function. Researchers have long taken advantage of the simple linear neuronal pathway, which begins at peripheral sensory modalities, travels through the central GF interneuron (GFI) to motor neurons, and terminates on wing/leg muscles. This circuit is more complex than it seems, however, as there exists a complex web of coupled neurons connected to the GFI that widely innervates the thoracic ganglion. Here, we define four new neuron clusters dye coupled to the central GFI, which we name GF coupled (GFC) 1–4. We identify new transgenic Gal4 drivers that express specifically in these neurons, and map both neuronal architecture and synaptic polarity. GFC1–4 share a central site of GFI connectivity, the inframedial bridge, where the neurons each form electrical synapses. Targeted apoptotic ablation of GFC1 reveals a key role for the proper development of the GF circuit, including the maintenance of GFI connectivity with upstream and downstream synaptic partners. GFC1 ablation frequently results in the loss of one GFI, which is always compensated for by contralateral innervation from a branch of the persisting GFI axon. Overall, this work reveals extensively coupled interconnectivity within the GF circuit, and the requirement of coupled neurons for circuit development. Identification of this large population of electrically coupled neurons in this classic model, and the ability to genetically manipulate these electrically synapsed neurons, expands the GF system capabilities for the nuanced, sophisticated circuit dissection necessary for deeper investigations into brain formation.

A Suite of Transgenic Driver and Reporter Mouse Lines with Enhanced Brain-Cell-Type Targeting and Functionality

Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

Multi-tissue GAL4-mediated gene expression in all Anopheles gambiae life stages using an endogenous polyubiquitin promoter

The ability to manipulate the Anopheles gambiae genome and alter gene expression effectively and reproducibly is a prerequisite for functional genetic analysis and for the development of novel control strategies in this important disease vector. However, in vivo transgenic analysis in mosquitoes is limited by the lack of promoters active ubiquitously. To address this, we used the GAL4/UAS system to investigate the promoter of the An. gambiae Polyubiquitin-c (PUBc) gene and demonstrated its ability to drive expression in mosquito cell culture before incorporation into An. gambiae transgenic driver lines. To generate such lines, piggyBac-mediated insertion was used to identify genomic regions able to sustain widespread expression and to create φC31 docking lines at these permissive sites. Patterns of expression induced by PUBc-GAL4 drivers carrying single intergenic insertions were assessed by crossing with a novel responder UAS-mCD8:mCherry line that was created by φC31-mediated integration. Amongst the drivers created at single, unique chromosomal integration loci, two were isolated that induced differential expression levels in a similar multiple-tissue spatial pattern throughout the mosquito life cycle. This work expands the tools available for An. gambiae functional analysis by providing a novel promoter for investigating phenotypes resulting from widespread multi-tissue expression, as well as identifying and tagging genomic sites that sustain broad transcriptional activity.

A Suite of Transgenic Driver and Reporter Mouse Lines with Enhanced Brain Cell Type Targeting and Functionality

Modern genetic approaches are powerful in providing access to diverse types of neurons within the mammalian brain and greatly facilitating the study of their function. We here report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically-defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of highprecision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.