Cre Driver Research Articles

Development of a novel immunocompetent murine tumor model for urothelial carcinoma using in vivo electroporation

A lack of advanced preclinical mouse tumor models impedes the progress in urothelial carcinoma research. We present here a novel fast, robust, reliable, and highly reproducible model for the genetic induction of bladder cancer in immunocompetent mice. Different sets of oncogenic transposons (Cmyc, Kras) and Cre drivers were transfected into the murine bladder wall of two different genetic backgrounds (Trp53fl/fl and BrafV600E, Ptenfl/fl, Ctnnb1exon3-fl/fl). Transfection was carried out using in vivo electroporation of the bladder after surgical exploration and transmural or transurethral intravesical plasmid injection. Up to 100% of animals developed urothelial carcinomas of the bladder. Time to tumor onset ranged from 16 to 97 days with a median of approximately 23 days in the fastest groups. Histological examination identified orthotopic urothelial carcinomas in most cases, in some experimental groups up to 100%. The resulting tumors were highly invasive and often metastatic. Metastases were found in up to 100% of tumor bearing mice per group. Taken together, this study establishes the proof-of-principle that in vivo electroporation can be versatilely employed as a reliable, fast, and robust method for the highly reproducible induction of urothelial carcinomas in the murine bladder wall. This novel murine tumor model could pave the way towards more easily modelling subtype specific urothelial carcinomas in mice.

Abstract A069: Development of an AAV-mediated GEM model to investigate the impact of age on pancreatic tumorigenesis

Abstract Much of our understanding of PDAC initiation comes from Genetically Engineered Mouse (GEM) models where a pancreas-specific Cre (such as Pdx- or p48-Cre) drives expression of specific mutations. While these GEM models generally recapitulate the key histopathological features of human PDAC, they have limitations when it comes to studying the impact of age on PDAC initiation since the Cre drivers induce recombination at the embryonic stage. This is an important consideration since in humans the disease manifests later in life, with the median age at diagnosis being 70 years old. Here, we describe the development of an intrapancreatic injection model using adeno-associated virus (AAV) to study how age influences PDAC initiation and the dynamics of tumor progression. This new viral transduction-based mouse model employs AAV-Cre to induce expression of an oncogenic KRasG12D allele together with a Trp53R172H tumor suppressor mutation within the pancreata of mice at different ages. Our approach utilizes mice with the genotype LSL-KrasG12D; LSL-Trp53R172H that are injected with different AAV-Cre directly into the parenchyma of the pancreas. In our initial studies, we used rAAV vectors expressing Cre recombinase and EGFP (AAV-Cre-EGFP) into young mice (10-14 weeks old) and older mice (32 weeks old) and analyzed EGFP expression using IVIS imaging and immunohistochemistry (IHC) at various timepoints post-injection. To evaluate tumor initiation, we assessed the extent of PanIN lesion formation using H&E staining. Our preliminary findings indicate that AAV-Cre-EGFP effectively transduces cells in vivo and triggers tumor development in the pancreas. To account for potential immunogenicity of the fluorescent protein, we then used rAAV vectors lacking EGFP expression. As controls, we administered identical rAAV vectors to wild-type cohorts matched for age and sex, along with mock injections. Transduction efficiency within the pancreas was determined by evaluating Cre and p53 expression levels, given that the R172H mutation stabilizes the protein. Notably, we found that EGFP is not necessary for either successful transduction or tumor initiation. We are currently evaluating PDAC initiation using a wide spectrum of ages, but our preliminary data using this model suggest that increased age may impact tumor progression, and that this effect may be sex-specific. Our novel AAV-Cre injection model holds promise for investigating age-related aspects of PDAC initiation, and in the future, such approaches could be leveraged to investigate how age impacts the effectiveness of different therapeutic modalities. Citation Format: Karen XD Duong-Polk, Madelaine Neff, Cosimo Commisso. Development of an AAV-mediated GEM model to investigate the impact of age on pancreatic tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A069.

Abstract B080: A porcine model of pancreatic ductal adenocarcinoma for human scale translational cancer research

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a highly fatal form of cancer, requiring urgent advancements in diagnostic and therapeutic strategies. Although PDAC mouse models have been crucial in advancing our comprehension of the disease, they present some limitations in replicating key human PDAC aspects. In this study, we present the development of the first large animal model of pancreatic cancer via Cre activatable mutations in the endogenous KRAS and TP53 genes 1,2. In order to activate these mutations in the pancreas, two Cre driver pig lines expressing Cre recombinase from PTF1A 3 and PDX1 were created. Crossing both Cre lines with pigs carrying latent KRAS G12D and TP53 R167H alleles produced double (KC) and triple (KPC) genetically modified animals. Pathological analyses revealed the presence of inter- and intralobular fibrosis with multifocal acinar-to-ductal metaplasia (ADM), some low-grade PanINs, dysplasia, inflammation and duct obstruction in most of the KPC pigs between 3 to 6 months of age. Interestingly, the majority of these pigs already exhibited local metastasis in the lymph nodes, suggesting that the metastatic potential is established early in the tumorigenic process. Age-matched KC pigs exhibited mainly ADM with dysplasia. Flow cytometry and RNA expression analyses of the established pancreatic tumour primary cells revealed a pronounced epithelial-to-mesenchymal transition (EMT). Moreover, both bulk tumour samples and primary tumour cells showed high expression of the pancreatic cancer stem cell marker CD44, with differential expression of its isoforms, including the major isoform and variants 6 and 9. In addition, histological and molecular analyses revealed notable similarities between pig and human PDAC, including molecular subtypes, neuronal invasion and loss of acinar identity. These findings highlight the unique advantage of the pig PDAC model, that allows longitudinal and multi-regional tumour sampling in the absence of any therapeutic interventions. This makes it a valuable tool for developing new diagnostic and therapeutic approaches that can greatly improve patient outcomes in the long run.

A safety guide for transgenic Cre drivers in metabolism.

A safety guide for transgenic Cre drivers in metabolism.

Deletion of the Ebf1, a mouse deafness gene, causes a dramatic increase in hair cells and support cells of the organ of Corti.

Following up on our previous observation that early B cell factor (EBF) sites are enriched in open chromatin of the developing sensory epithelium of the mouse cochlea, we investigated the effect of deletion of Ebf1 on inner ear development. We used a Cre driver to delete Ebf1 at the otocyst stage before development of the cochlea. We examined the cochlea at postnatal day (P) 1 and found that the sensory epithelium had doubled in size but the length of the cochlear duct was unaffected. We also found that deletion of Ebf1 led to ectopic sensory patches in the Kölliker's organ. Innervation of the developing organ of Corti was disrupted with no obvious spiral bundles. The ectopic patches were also innervated. All the extra hair cells (HCs) within the sensory epithelium and Kölliker's organ contained mechanoelectrical transduction channels, as indicated by rapid uptake of FM1-43. The excessive numbers of HCs were still present in the adult Ebf1 conditional knockout (cKO) animal. The animals had significantly elevated auditory brainstem response thresholds, suggesting that this gene is essential for hearing development.

Sensory-motor circuit is a therapeutic target for dystonia musculorum mice, a model of hereditary sensory and autonomic neuropathy 6.

Mutations in Dystonin (DST), which encodes cytoskeletal linker proteins, cause hereditary sensory and autonomic neuropathy 6 (HSAN-VI) in humans and the dystonia musculorum (dt) phenotype in mice; however, the neuronal circuit underlying the HSAN-VI and dt phenotype is unresolved. dt mice exhibit dystonic movements accompanied by the simultaneous contraction of agonist and antagonist muscles and postnatal lethality. Here, we identified the sensory-motor circuit as a major causative neural circuit using a gene trap system that enables neural circuit-selective inactivation and restoration of Dst by Cre-mediated recombination. Sensory neuron-selective Dst deletion led to motor impairment, degeneration of proprioceptive sensory neurons, and disruption of the sensory-motor circuit. Restoration of Dst expression in sensory neurons using Cre driver mice or a single postnatal injection of Cre-expressing adeno-associated virus ameliorated sensory degeneration and improved abnormal movements. These findings demonstrate that the sensory-motor circuit is involved in the movement disorders in dt mice and that the sensory circuit is a therapeutic target for HSAN-VI.

B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

B cells and the antibodies they produce are critical in host defense against pathogens and contribute to various immune-mediated diseases. B cells responding to activating signals in vitro release extracellular vesicles (EV) that carry surface antibodies, yet B cell production of EVs that express antibodies and their function in vivo is incompletely understood. Using transgenic mice expressing the Cre recombinase in B cells switching to IgG1 to induce expression of fusion proteins between emerald green fluorescent protein (emGFP) and the EV tetraspanin CD63 as a model, we identify emGFP expression in B cells responding to foreign antigen in vivo and characterize the emGFP+ EVs they release. Our data suggests that emGFP+ germinal center B cells undergoing immunoglobulin class switching to express IgG and their progeny memory B cells and plasma cells, also emGFP+, are sources of circulating antigen-specific IgG+ EVs. Furthermore, using a mouse model of influenza virus infection, we find that IgG+ EVs specific for the influenza hemagglutinin antigen protect against virus infection. In addition, crossing the B cell Cre driver EV reporter mice onto the Nba2 lupus-prone strain revealed increased circulating emGFP+ EVs that expressed surface IgG against nuclear antigens linked to autoimmunity. These data identify EVs loaded with antibodies as a novel route for antibody secretion in B cells that contribute to adaptive immune responses, with important implications for different functions of IgG+ EVs in infection and autoimmunity.

Dopamine production in neurotensin receptor 1 neurons is required for diet-induced obesity and increased day eating on a high-fat diet.

This study aimed to determine a dopaminergic circuit required for diet-induced obesity in mice. We created conditional deletion mutants for tyrosine hydroxylase (TH) using neurotensin receptor 1 (Ntsr1) Cre and other Cre drivers and measured feeding and body weight on standard and high-fat diets. We then used an adeno-associated virus to selectively restore TH to the ventral tegmental area (VTA) Ntsr1 neurons in conditional knockout (cKO) mice. Mice with cKO of Th using Vglut2-Cre, Cck-Cre, Calb1-Cre, and Bdnf-Cre were susceptible to obesity on a high-fat diet; however, Ntsr1-Cre Th cKO mice resisted weight gain on a high-fat diet and did not experience an increase in day eating unlike their wild-type littermate controls. Restoration of TH to the VTA Ntsr1 neurons of the Ntsr1-Cre Th cKO mice using an adeno-associated virus resulted in an increase in weight gain and day eating on a high-fat diet. Ntsr1-Cre Th cKO mice failed to increase day eating on a high-fat diet, offering a possible explanation for their resistance to diet-induced obesity. These results implicate VTA Ntsr1 dopamine neurons as promoting out-of-phase feeding behavior on a high-fat diet that could be an important contributor to diet-induced obesity in humans.

Transgenic Targeting of Fcrls Creates a Highly Efficient Constitutively Active Microglia Cre Line with Differentiated Specificity.

Microglia carry out important functions as the resident macrophages of the brain. To study their role in health and disease, the research community needs tools to genetically modify them with maximum completeness in a manner that distinguishes them from closely related cell types, such as monocytes. While currently available tamoxifen-inducible CreERT2 lines can achieve the differentiation from other cells, the field needs improved and publicly available constitutively active Cre lines, especially ones with favorable efficiency and specificity profiles for studies where high recombination efficiency is imperative and where tamoxifen administration is contraindicated. Here, we leverage the microglia-specific Fcrls gene to generate mice expressing Cre. Using genomic methods, we show correct positioning of the transgene and intact microglia homeostasis in Fcrls-2A-Cre mice. Crossing Fcrls-2A-Cre mice to four different reporters, we demonstrate highly efficient recombination in microglia across differentially sensitive loxP alleles in different genomic contexts, indicating robust applicability of the line. Further, we show that microglia recombine a loxP reporter during early embryonic development, supporting the use of the line for developmental studies. Finally, using immunofluorescence and flow cytometry, we reveal that most border-associated macrophages are also targeted whereas only few liver and spleen macrophages and virtually no white blood cell subsets exhibit Cre activity, distinguishing this line from another publicly available Cre line, Cx3cr1-CreM Fcrls-2A-Cre mice are immediately available (JAX #036591) and serve as a valuable addition to the community's microglia toolbox by providing highly efficient constitutive Cre activity with excellent specificity, particularly for studies where tamoxifen administration is undesirable.

Injury-induced Foxm1 expression in the mouse kidney drives epithelial proliferation by a cyclin F-dependent mechanism.

Acute kidney injury (AKI) strongly upregulates the transcription factor Foxm1 in the proximal tubule in vivo, and Foxm1 drives epithelial proliferation in vitro. Here, we report that deletion of Foxm1 either with a nephron-specific Cre driver or by inducible global deletion reduced proximal tubule proliferation after ischemic injury in vivo. Foxm1 deletion led to increased AKI to chronic kidney disease transition, with enhanced fibrosis and ongoing tubule injury 6 weeks after injury. We report ERK mediated FOXM1 induction downstream of the EGFR in primary proximal tubule cells. We defined FOXM1 genomic binding sites by cleavage under targets and release using nuclease (CUT&RUN) and compared the genes located near FOXM1 binding sites with genes downregulated in primary proximal tubule cells after FOXM1 knockdown. The aligned data sets revealed the cell cycle regulator cyclin F (CCNF) as a putative FOXM1 target. We identified 2 cis regulatory elements that bound FOXM1 and regulated CCNF expression, demonstrating that Ccnf is strongly induced after kidney injury and that Foxm1 deletion abrogates Ccnf expression in vivo and in vitro. Knockdown of CCNF also reduced proximal tubule proliferation in vitro. These studies identify an ERK/FOXM1/CCNF signaling pathway that regulates injury-induced proximal tubule cell proliferation.

An inducible genetic tool to track and manipulate specific microglial states reveals their plasticity and roles in remyelination

Recent single-cell RNA sequencing studies have revealed distinct microglial states in development and disease. These include proliferative-region-associated microglia (PAMs) in developing white matter and disease-associated microglia (DAMs) prevalent in various neurodegenerative conditions. PAMs and DAMs share a similar core gene signature. However, the extent of the dynamism and plasticity of these microglial states, as well as their functional significance, remains elusive, partly due to the lack of specific tools. Here, we generated an inducible Cre driver line, Clec7a-CreERT2, that targets PAMs and DAMs in the brain parenchyma.Utilizing this tool, we profiled labeled cells during development and in several disease models, uncovering convergence and context-dependent differences in PAM and DAM gene expression. Through long-term tracking, we demonstrated microglial state plasticity. Lastly, we specifically depleted DAMs in demyelination, revealing their roles in disease recovery. Together, we provide a versatile genetic tool to characterize microglial states in CNS development and disease.

Colonic MYO5B Expression Retains Fecal Formation but not Weight Loss in OLFM4+ Stem Cell-Specific MYO5B Deletion in Mice

Background and aim: Microvillus inclusion disease (MVID) is a congenital disorder characterized by severe diarrhea and is caused by inactivating mutations in motor protein myosin Vb (MYO5B). Mice lacking MYO5B in intestinal and colonic epithelia ( Vil-CreERT2;Myo5bfl/fl) display an MVID phenotype characterized by significant weight loss, lack of solid feces, and brush border defects. However, previous models have not discerned if this diarrheal phenotype is due to MYO5B loss in the small intestine specifically or both in the small intestine and colon. Using the small intestinal stem cell specific Cre driver under Olfm4, we phenotyped the mice lacking MYO5B only in the small intestine, while normal MYO5B expression is maintained in the colon. Methods: Olfm4-GFP-CreERT2;R26R-LSL-tdTom and Myo5bfl/fl mice were crossbred to generate Olfm4-GFP-CreERT2;R26R-LSL-tdTom;Myo5bfl/fl (Olfm4DMYO5B) mice. Adult Olfm4DMYO5B mice and control littermates received tamoxifen injection at day 0. GI tissues were collected 5 and 7 days post tamoxifen. Results: Following Cre induction, Olfm4DMYO5B mice exhibited progressive weight loss starting at day 5 and on average lost 20.6% on day 7. At day 7, the small intestine contained clear luminal contents, similar to that of the previous MVID model mice. However, the colon of day 7 Olfm4DMYO5B mice contained small, solid feces. An elongation of PCNA+ crypts was identified on day 5 throughout the small intestine. Villus blunting and microvillus inclusions were prominent in the small intestinal enterocytes on day 7, but not on day 5. The day 7 Olfm4DMYO5B small intestine demonstrated a decrease in tuft cell numbers, suggesting differentiation deficits in the MYO5B-lacking stem cells. However, the colon of Olfm4DMYO5B mice showed intact morphology. Colonic crypt depth and tuft cell numbers at both days 5 and 7 were similar to those of control mice. Immunostaining confirmed the maintenance of MYO5B expression in the colon and progressive loss of MYO5B in the small intestine of Olfm4DMYO5B mice. At day 5, MYO5B was specifically lost in small intestinal crypts, and the loss of MYO5B expanded to the villus tips on day 7. These MYO5B-lacking cells were tdTom+, including enterocytes, goblet, and rare tuft cells. Following the loss of MYO5B, small intestinal enterocytes lacked proper apical localization of numerous transporters and enzymes, including Na+-glucose cotransporter 1 and dipeptidyl peptidase 4. Conclusion: Recently established Olfm4DMYO5B mice demonstrated that the specific MYO5B deletion in the small intestinal stem cells causes hyperproliferation and differentiation deficits. Colonic MYO5B function is suffcient for the formation of solid feces, however, these mice experience prominent weight loss, likely due to the MVID-like malabsorption in the small intestine. NIH R01DK128190, RC2DK118640. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Development and characterization of a Gucy2d-cre mouse to selectively manipulate a subset of inhibitory spinal dorsal horn interneurons.

Recent transcriptomic studies identified Gucy2d (encoding guanylate cyclase D) as a highly enriched gene within inhibitory dynorphin interneurons in the mouse spinal dorsal horn. To facilitate investigations into the role of the Gucy2d+ population in somatosensation, Gucy2d-cre transgenic mice were created to permit chemogenetic or optogenetic manipulation of this subset of spinal neurons. Gucy2d-cre mice created via CRISPR/Cas9 genomic knock-in were bred to mice expressing a cre-dependent reporter (either tdTomato or Sun1.GFP fusion protein), and the resulting offspring were characterized. Surprisingly, a much wider population of spinal neurons was labeled by cre-dependent reporter expression than previous mRNA-based studies would suggest. Although the cre-dependent reporter expression faithfully labeled ~75% of cells expressing Gucy2d mRNA in the adult dorsal horn, it also labeled a substantial number of additional inhibitory neurons in which no Gucy2d or Pdyn mRNA was detected. Moreover, cre-dependent reporter was also expressed in various regions of the brain, including the spinal trigeminal nucleus, cerebellum, thalamus, somatosensory cortex, and anterior cingulate cortex. Injection of AAV-CAG-FLEX-tdTomato viral vector into adult Gucy2d-cre mice produced a similar pattern of cre-dependent reporter expression in the spinal cord and brain, which excludes the possibility that the unexpected reporter-labeling of cells in the deep dorsal horn and brain was due to transient Gucy2d expression during early stages of development. Collectively, these results suggest that Gucy2d is expressed in a wider population of cells than previously thought, albeit at levels low enough to avoid detection with commonly used mRNA-based assays. Therefore, it is unlikely that these Gucy2d-cre mice will permit selective manipulation of inhibitory signaling mediated by spinal dynorphin interneurons, but this novel cre driver line may nevertheless be useful to target a broader population of inhibitory spinal dorsal horn neurons.

Abstract PR-010: Asc1lb progenitor-specific RB conditional inactivation in zebrafish models rare CNS primitive neuroectodermal tumors

Abstract PR-010: Asc1lb progenitor-specific RB conditional inactivation in zebrafish models rare CNS primitive neuroectodermal tumors

Carotid Body Function in Tyrosine Hydroxylase Conditional Olfr78 Knockout Mice.

The Olfr78 gene encodes a G-protein-coupled olfactory receptor that is expressed in several ectopic sites. Olfr78 is one of the most abundant mRNA species in carotid body (CB) glomus cells. These cells are the prototypical oxygen (O2) sensitive arterial chemoreceptors, which, in response to lowered O2 tension (hypoxia), activate the respiratory centers to induce hyperventilation. It has been proposed that Olfr78 is a lactate receptor and that glomus cell activation by the increase in blood lactate mediates the hypoxic ventilatory response (HVR). However, this proposal has been challenged by several groups showing that Olfr78 is not a physiologically relevant lactate receptor and that the O2-based regulation of breathing is not affected in constitutive Olfr78 knockout mice. In another study, constitutive Olfr78 knockout mice were reported to have altered systemic and CB responses to mild hypoxia. To further characterize the functional role of Olfr78 in CB glomus cells, we here generated a conditional Olfr78 knockout mouse strain and then restricted the knockout to glomus cells and other catecholaminergic cells by crossing with a tyrosine hydroxylase-specific Cre driver strain (TH-Olfr78 KO mice). We find that TH-Olfr78 KO mice have a normal HVR. Interestingly, glomus cells of TH-Olfr78 KO mice exhibit molecular and electrophysiological alterations as well as a reduced dopamine content in secretory vesicles and neurosecretory activity. These functional characteristics resemble those of CB neuroblasts in wild-type mice. We suggest that, although Olfr78 is not essential for CB O2 sensing, activation of Olfr78-dependent pathways is required for maturation of glomus cells.

Metabolic and behavioral alterations associated with viral vector-mediated toxicity in the paraventricular hypothalamic nucleus.

Combining adeno-associated virus (AAV)-mediated expression of Cre recombinase with genetically modified floxed animals is a powerful approach for assaying the functional role of genes in regulating behavior and metabolism. Extensive research in diverse cell types and tissues using AAV-Cre has shown it can save time and avoid developmental compensation as compared to using Cre driver mouse line crossings. We initially sought to study the impact of ablation of corticotropin-releasing hormone (CRH) in the paraventricular hypothalamic nucleus (PVN) using intracranial AAV-Cre injection in adult animals. In this study, we stereotactically injected AAV8-hSyn-Cre or a control AAV8-hSyn-GFP both Crh-floxed and wild-type mouse PVN to assess behavioral and metabolic impacts. We then used immunohistochemical markers to systematically evaluate the density of hypothalamic peptidergic neurons and glial cells. We found that delivery of one specific preparation of AAV8-hSyn-Cre in the PVN led to the development of obesity, hyperphagia, and anxiety-like behaviors. This effect occurred independent of sex and in both floxed and wild-type mice. We subsequently found that AAV8-hSyn-Cre led to neuronal cell death and gliosis at the site of viral vector injections. These behavioral and metabolic deficits were dependent on injection into the PVN. An alternatively sourced AAV-Cre did not reproduce the same results. Our findings reveal that delivery of a specific batch of AAV-Cre could lead to cellular toxicity and lesions in the PVN that cause robust metabolic and behavioral impacts. These alterations can complicate the interpretation of Cre-mediated gene knockout and highlight the need for rigorous controls.

New Flexon-based reagents for tissue-specific Auxin-Inducible Degradation and for characterizing Cre and Flp drivers in C. elegans.

A Flexon stop cassette interrupts translation of a coding region until it is excised by a recombinase to allow for gene expression. We have expanded options for Auxin-Inducible Degradation by generating Flexon-based transgenes for tissue-specific expression of the ubiquitin ligase substrate recognition component TIR1 or the variant TIR1(F79G) after excision of the Flexon by Cre recombinase. We also describe Flexon-based tester transgenes to facilitate gathering accurate information about the expression pattern of Cre and Flp recombinase drivers that can be used in conjunction with any conditional expression reagents that utilize these recombinases.

From Individual to Population: Circuit Organization of Pyramidal Tract and Intratelencephalic Neurons in Mouse Sensorimotor Cortex.

The sensorimotor cortex participates in diverse functions with different reciprocally connected subregions and projection-defined pyramidal neuron types therein, while the fundamental organizational logic of its circuit elements at the single-cell level is still largely unclear. Here, using mouse Cre driver lines and high-resolution whole-brain imaging to selectively trace the axons and dendrites of cortical pyramidal tract (PT) and intratelencephalic (IT) neurons, we reconstructed the complete morphology of 1,023 pyramidal neurons and generated a projectome of 6 subregions within the sensorimotor cortex. Our morphological data revealed substantial hierarchical and layer differences in the axonal innervation patterns of pyramidal neurons. We found that neurons located in the medial motor cortex had more diverse projection patterns than those in the lateral motor and sensory cortices. The morphological characteristics of IT neurons in layer 5 were more complex than those in layer 2/3. Furthermore, the soma location and morphological characteristics of individual neurons exhibited topographic correspondence. Different subregions and layers were composed of different proportions of projection subtypes that innervate downstream areas differentially. While the axonal terminals of PT neuronal population in each cortical subregion were distributed in specific subdomains of the superior colliculus (SC) and zona incerta (ZI), single neurons selectively innervated a combination of these projection targets. Overall, our data provide a comprehensive list of projection types of pyramidal neurons in the sensorimotor cortex and begin to unveil the organizational principle of these projection types in different subregions and layers.

Col1α2-Cre-mediated recombination occurs in various cell types due to Cre expression in epiblasts

The Cre-LoxP system has been commonly used for cell-specific genetic manipulation. However, many Cre strains exhibit excision activity in unexpected cell types or tissues. Therefore, it is important to identify the cell types in which recombination takes place. Fibroblasts are a cell type that is inadequately defined due to a lack of specific markers to detect the entire cell lineage. Here, we investigated the Cre recombination induced by Col1α2-iCre, one of the most common fibroblast-mesenchymal Cre driver lines, by using a double-fluorescent Cre reporter line in which GFP is expressed when recombination occurs. Our results indicated that Col1α2-iCre activity was more extensive across cell types than previously reported: Col1α2-iCre-mediated recombination was found in not only cells of mesenchymal origin but also those of other lineages, including haematopoietic cells, myocardial cells, lung and intestinal epithelial cells, and neural cells. In addition, study of embryos revealed that recombination by Col1α2-iCre was observed in the early developmental stage before gastrulation in epiblasts, which would account for the recombination across various cell types in adult mice. These results offer more insights into the activity of Col1α2-iCre and suggest that experimental results obtained using Col1α2-iCre should be carefully interpreted.