Cre-driver Mouse Research Articles

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.

Large-Scale Genome-Wide Optimization and Prediction of the Cre Recombinase System for Precise Genome Manipulation in Mice.

The Cre-Lox recombination system is a powerful tool in mouse genetics, offering spatial-temporal control over gene expression and facilitating the large-scale generation of conditional knockout mice. Its versatility also extends to other research models, such as rats, pigs, and zebrafish. However, the Cre-Lox technology presents a set of challenges that includes high costs, a time-intensive process, and the occurrence of unpredictable recombination events, which can lead to unexpected phenotypic outcomes. To better understand factors affecting recombination, we embarked on a systematic and genome-wide analysis of Cre-mediated recombination in mice. To ensure uniformity and reproducibility, we generated 11 novel strains with conditional alleles at the ROSA26 locus, utilizing a single inbred mouse strain background, C57BL/6J. We examined several factors influencing Cre-recombination, including the inter-loxP distance, mutant loxP sites, the zygosity of the conditional alleles, chromosomal location, and the age of the breeders. We discovered that the selection of the Cre-driver strain profoundly impacts recombination efficiency. We also found that successful and complete recombination is best achieved when loxP sites are spaced between 1 to 4 kb apart, with mutant loxP sites facilitating recombination at distances of 1 to 3 kb. Furthermore, we demonstrate that complete recombination does not occur at an inter-loxP distance of ≥ 15 kb with wildtype loxP sites, nor at a distance of ≥ 7 kb with mutant lox71/66 sites. Interestingly, the age of the Cre-driver mouse at the time of breeding emerged as a critical factor in recombination efficiency, with best results observed between 8 and 20 weeks old. Moreover, crossing heterozygous floxed alleles with the Cre-driver strain resulted in more efficient recombination than using homozygous floxed alleles. Lastly, maintaining an inter-loxP distance of 4 kb or less ensures efficient recombination of the conditional allele, regardless of the chromosomal location. While CRISPR/Cas has revolutionized genome editing in mice, Cre-Lox technology remains a cornerstone for the generation of sophisticated alleles and for precise control of gene expression in mice. The knowledge gained here will enable investigators to select a Cre-Lox approach that is most efficient for their desired outcome in the generation of both germline and non-germline mouse models of human disease, thereby reducing time and cost of Cre-Lox technology-mediated genome modification.

Large-Scale Genome-Wide Optimization and Prediction of the Cre Recombinase System for Precise Genome Manipulation in Mice.

The Cre-Lox recombination system is a powerful tool in mouse genetics, offering spatial-temporal control over gene expression and facilitating the large-scale generation of conditional knockout mice. Its versatility also extends to other research models, such as rats, pigs, and zebrafish. However, the Cre-Lox technology presents a set of challenges that includes high costs, a time-intensive process, and the occurrence of unpredictable recombination events, which can lead to unexpected phenotypic outcomes. To better understand factors affecting recombination, we embarked on a systematic and genome-wide analysis of Cre-mediated recombination in mice. To ensure uniformity and reproducibility, we generated 11 novel strains with conditional alleles at the ROSA26 locus, utilizing a single inbred mouse strain background, C57BL/6J. We examined several factors influencing Cre-recombination, including the inter-loxP distance, mutant loxP sites, the zygosity of the conditional alleles, chromosomal location, and the age of the breeders. We discovered that the selection of the Cre-driver strain profoundly impacts recombination efficiency. We also found that successful and complete recombination is best achieved when loxP sites are spaced between 1 to 4 kb apart, with mutant loxP sites facilitating recombination at distances of 1 to 3 kb. Furthermore, we demonstrate that complete recombination does not occur at an inter-loxP distance of ≥ 15 kb with wildtype loxP sites, nor at a distance of ≥ 7 kb with mutant lox71/66 sites. Interestingly, the age of the Cre-driver mouse at the time of breeding emerged as a critical factor in recombination efficiency, with best results observed between 8 and 20 weeks old. Moreover, crossing heterozygous floxed alleles with the Cre-driver strain resulted in more efficient recombination than using homozygous floxed alleles. Lastly, maintaining an inter-loxP distance of 4 kb or less ensures efficient recombination of the conditional allele, regardless of the chromosomal location. While CRISPR/Cas has revolutionized genome editing in mice, Cre-Lox technology remains a cornerstone for the generation of sophisticated alleles and for precise control of gene expression in mice. The knowledge gained here will enable investigators to select a Cre-Lox approach that is most efficient for their desired outcome in the generation of both germline and non-germline mouse models of human disease, thereby reducing time and cost of Cre-Lox technology-mediated genome modification.

Interplay between Pitx2 and Pax7 temporally governs specification of extraocular muscle stem cells.

Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression. We showed that this gene becomes progressively dispensable for specification and maintenance of the muscle stem (MuSC) cell pool in extraocular muscles (EOMs) despite being, together with Myf5, a major upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally led to a greater loss of MuSCs in the EOMs compared to the limb. Thus, we propose a relay between Pitx2, Myf5 and Pax7 for EOM stem cell maintenance. We demonstrate also that MuSCs in the EOMs adopt a quiescent state earlier that those in limb muscles and do not spontaneously proliferate in the adult, yet EOMs have a significantly higher content of Pax7+ MuSCs per area pre- and post-natally. Finally, while limb MuSCs proliferate in the mdx mouse model for Duchenne muscular dystrophy, significantly less MuSCs were present in the EOMs of the mdx mouse model compared to controls, and they were not proliferative. Overall, our study provides a comprehensive in vivo characterisation of MuSC heterogeneity along the body axis and brings further insights into the unusual sparing of EOMs during muscular dystrophy.

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.

Distinct distribution of subplate neuron subtypes between the sensory cortices during the early postnatal period.

Subplate neurons (SpNs) are a heterogeneous neuronal population actively involved in early cortical circuit formation. In rodents, many SpNs survive and form layer 6b. The molecular heterogeneity of SpNs raises the question of whether different subpopulations of SpNs survive through the early postnatal period similarly and whether such diverse SpN populations in the auditory cortex(ACtx) share a common distribution pattern with other sensory systems. To address that, we investigated the expression pattern of multiple specific SpN markers in the ACtx, as well as in the visual (VCtx) and somatosensory (SCtx) cortices as controls, using complexin 3 (Cplx3) antibodies and different SpN-specific Cre-driver mice, such as connective tissue growth factor (CTGF), dopamine receptor D1 (Drd1a), and neurexophilin 4 (Nxph4). We focused on two early time windows in auditory development: (1) during the second postnatal week (PNW) before ear-canal opening and (2) during the third PNW after ear-canal opening. We compared the expression pattern of different SpN markers in ACtx with VCtx and SCtx. At both examined timepoints, Cplx3 and Nxph4 expressing SpNs form the largest and smallest population in the ACtx, respectively. Similar distribution patterns are observable in the VCtx and SCtx during the second PNW but not during the third PNW, for a higher proportion of Drd1a expressing SpNs is detected in the VCtx and CTGF expressing SpNs in the SCtx. This study suggests that different populations of SpNs might contribute differently to the development of individual sensory circuits.

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.

Variegate expression of Cre recombinase in hematopoietic cells in CD11c-cre transgenic mice

In this study, we performed an in-depth analysis of Cre expression in the widely used CD11c-Cre transgenic mice generated by the group of Boris Reizis. In contrast to previous observation, using the highly sensitive Rosa-26-floxed-tdTomato reporter mouse line, we show variegated expression of Cre in multiple hematopoietic linage cells starting in hematopoietic stem cells. Indeed, we found that in the CD11c-Cre driver mice, Cre is expressed in cDC linage cells and pDC starting from the myeloid dendritic cell precursor, as expected, but also in a substantial fraction of hematopoietic stem cells and common lymphoid progenitors and, consequently, in >50% of all leukocytes. Hence, this study indicates that the reporter mice used to characterize Cre expression in Cre-driver mice should be selected with caution and considering the sensitivity of the reporter system. This study also suggests that the interpretation of some reports using this CD11c-Cre transgenic mice may need to be re-considered based on a careful evaluation of the cell type-specificity of Cre-mediated in their model.

A Versatile Strategy for Genetic Manipulation of Cajal-Retzius Cells in the Adult Mouse Hippocampus.

Cajal-Retzius (CR) cells are transient neurons with long-lasting effects on the architecture and circuitry of the neocortex and hippocampus. Contrary to the prevailing assumption that CR cells completely disappear in rodents shortly after birth, a substantial portion of these cells persist in the hippocampus throughout adulthood. The role of these surviving CR cells in the adult hippocampus is largely unknown, partly because of the paucity of suitable tools to dissect their functions in the adult versus the embryonic brain. Here, we show that genetic crosses of the ΔNp73-Cre mouse line, widely used to target CR cells, to reporter mice induce reporter expression not only in CR cells, but also progressively in postnatal dentate gyrus granule neurons. Such a lack of specificity may confound studies of CR cell function in the adult hippocampus. To overcome this, we devise a method that not only leverages the temporary CR cell-targeting specificity of the ΔNp73-Cre mice before the first postnatal week, but also capitalizes on the simplicity and effectiveness of freehand neonatal intracerebroventricular injection of adeno-associated virus. We achieve robust Cre-mediated recombination that remains largely restricted to hippocampal CR cells from early postnatal age to adulthood. We further demonstrate the utility of this method to manipulate neuronal activity of CR cells in the adult hippocampus. This versatile and scalable strategy will facilitate experiments of CR cell-specific gene knockdown and/or overexpression, lineage tracing, and neural activity modulation in the postnatal and adult brain.

Development of two mouse strains conditionally expressing bright luciferases with distinct emission spectra as new tools for in vivo imaging.

In vivo bioluminescence imaging (BLI) has been an invaluable noninvasive method to visualize molecular and cellular behaviors in laboratory animals. Bioluminescent reporter mice harboring luciferases for general use have been limited to a classical luciferase, Luc2, from Photinus pyralis, and have been extremely powerful for various in vivo studies. However, applicability of reporter mice for in vivo BLI could be further accelerated by increasing light intensity through the use of other luciferases and/or by improving the biodistribution of their substrates in the animal body. Here we created two Cre-dependent reporter mice incorporating luciferases oFluc derived from Pyrocoeli matsumurai and Akaluc, both of which had been reported previously to be brighter than Luc2 when using appropriate substrates; we then tested their bioluminescence in neural tissues and other organs in living mice. When expressed throughout the body, both luciferases emitted an intense yellow (oFluc) or far-red (Akaluc) light easily visible to the naked eye. oFluc and Akaluc were similarly bright in the pancreas for in vivo BLI; however, Akaluc was superior to oFluc for brain imaging, because its substrate, AkaLumine-HCl, was distributed to the brain more efficiently than the oFluc substrate, D-luciferin. We also demonstrated that the lights produced by oFluc and Akaluc were sufficiently spectrally distinct from each other for dual-color imaging in a single living mouse. Taken together, these novel bioluminescent reporter mice are an ideal source of cells with bright bioluminescence and may facilitate in vivo BLI of various tissues/organs for preclinical and biomedical research in combination with a wide variety of Cre-driver mice.

Abstract P3020: O-GlcNAc Transferase Regulates Vascular Smooth Muscle Cell Cycle Progression In Diabetes

Diabetic patients show increased proclivity for vascular smooth muscle cell (VSMC) migration and proliferation. Hyperglycemia, hallmark of diabetes, increases intracellular glucose signaling via O-linked N-acetylglucosamine transferase (OGT), a key regulator of protein O-GlcNAcylation. We previously showed that smooth muscle OGT (smOGT) deletion prevents hyperglycemia-induced atherosclerosis. Goal of this work was to interrogate the role of smOGT in VSMC cell cycle progression in diabetes. For this, we used conditional SMC-specific OGT knockout (smOGT KO ) and wild-type (smOGT WT ) mice generated via tamoxifen (tmx)-inducible VSMC-restricted Cre driver mice ( Itga8-CreER T2 ). Briefly, following tmx injection, 8-wks-old smOGT KO and smOGT WT mice were placed on Western diet feeding regimen for study duration. At 14-wks-age, mice were injected with 50mg/Kg/day streptozotocin i.p. once daily for 5 consecutive days. Mice were harvested at 16-18-wks-age and plasma and aortic tissues were collected for biochemical and molecular studies. Immunoblotting of aortic lysates revealed a significant decrease (59%) in OGT and O-GlcNAc expression, validating the smOGT KO mice. Loss of smOGT reduced (40%) CDK6 expression (regulator of G1-S phase cell cycle progression) concomitant to enhanced (62%) p21 Cip1 expression (cyclin-dependent kinase inhibitor) in aortic vessels from diabetic smOGT KO vs. smOGT WT mice. This was further accompanied with 69% attenuation in SRF expression (transcriptional regulator of SM proliferation) in diabetic smOGT KO vs. smOGT WT aortic vessels. Similar results were obtained with OGT loss-of-function studies in human coronary artery SMC (HCASMC) primary cultures under diabetic conditions in vitro . Briefly, control (siCtrl) and OGT siRNA (siOGT)-transfected HCASMC were treated with or without 25mM glucose+100μM PUGNAc for 48 hours. Immunoblotting revealed that reduced OGT-mediated O-GlcNAcylation in siOGT cells in a diabetic milieu significantly attenuated SRF expression coupled with augmented p21 Cip1 expression compared to diabetic siCtrl cells. Together, our results suggest a direct regulatory role of smooth muscle OGT in VSMC cell cycle progression in diabetes.

Generation of bicistronic Dmp1-Cre knock-in mice using a self-cleaving 2A peptide.

The conditional manipulation of genes using the Cre recombinase-locus of crossover in P1 (Cre/loxP) system is an important tool for revealing gene functions and cell lineages in vivo. The outcome of this method is dependent on the performance of Cre-driver mouse strains. In most cases, Cre knock-in mice show better specificity than randomly inserted Cre transgenic mice. However, following knock-in, the expression of the original gene replaced by Cre is lost. We generated a new differentiated osteoblast- and osteocyte-specific Cre knock-in mouse line that carries the viral T2A sequence encoding a 2A self-cleaving peptide at the end of the coding region of the dentin matrix protein 1 (Dmp1) gene accompanied by the Cre gene. We confirmed that Dmp1-T2A-Cre mice showed high Cre expression in osteoblasts, osteocytes, odontoblasts, and periodontal ligament cells and that the 2A self-cleaving peptide efficiently produced both Dmp1 and Cre proteins. Furthermore, unlike the Dmp1 knockout mice, homozygous Dmp1-T2A-Cre mice showed no skeletal abnormalities. Analysis using the Cre reporter strain confirmed differentiated osteoblast- and osteocyte-specific Cre-mediated recombination in the skeleton. Furthermore, recombination was also detected in some nuclei of skeletal muscle cells, spermatocytes, and intestinal cells. 2A-Cre functions effectively in vivo, and Dmp1-T2A-Cre knock-in mice are a useful tool for studying the functioning of various genes in hard tissues.

Cre-dependent ACR2-expressing reporter mouse strain for efficient long-lasting inhibition of neuronal activity

Optogenetics is a powerful tool for manipulating neuronal activity by light illumination with high temporal and spatial resolution. Anion-channelrhodopsins (ACRs) are light-gated anion channels that allow researchers to efficiently inhibit neuronal activity. A blue light-sensitive ACR2 has recently been used in several in vivo studies; however, the reporter mouse strain expressing ACR2 has not yet been reported. Here, we generated a new reporter mouse strain, LSL-ACR2, in which ACR2 is expressed under the control of Cre recombinase. We crossed this strain with a noradrenergic neuron-specific driver mouse (NAT-Cre) to generate NAT-ACR2 mice. We confirmed Cre-dependent expression and function of ACR2 in the targeted neurons by immunohistochemistry and electrophysiological recordings in vitro, and confirmed physiological function using an in vivo behavioral experiment. Our results show that the LSL-ACR2 mouse strain can be applied for optogenetic inhibition of targeted neurons, particularly for long-lasting continuous inhibition, upon crossing with Cre-driver mouse strains. The LSL-ACR2 strain can be used to prepare transgenic mice with homogenous expression of ACR2 in targeted neurons with a high penetration ratio, good reproducibility, and no tissue invasion.

Periderm Fate during Palatogenesis: TGF-β and Periderm Dedifferentiation

Failure of palatogenesis results in cleft palate, one of the most common congenital disabilities in humans. During the final phases of palatogenesis, the protective function of the peridermal cell layer must be eliminated for the medial edge epithelia to adhere properly, which is a prerequisite for the successful fusion of the secondary palate. However, a deeper understanding of the role and fate of the periderm in palatal adherence and fusion has been hampered due to a lack of appropriate periderm-specific genetic tools to examine this cell type in vivo. Here we used the cytokeratin-6A (Krt-6a) locus to develop both constitutive (Krt6ai-Cre) and inducible (Krt6ai-Cre ERT2 ) periderm-specific Cre driver mouse lines. These novel lines allowed us to achieve both the spatial and temporal control needed to dissect the periderm fate on a cellular resolution during palatogenesis. Our studies suggest that, already before the opposing palatal shelves contact each other, at least some palatal periderm cells start to gradually lose their squamous periderm-like phenotype and dedifferentiate into cuboidal cells, reminiscent of the basal epithelial cells seen in the palatal midline seam. Moreover, we show that transforming growth factor–β (TGF-β) signaling plays a critical periderm-specific role in palatogenesis. Thirty-three percent of embryos lacking a gene encoding the TGF-β type I receptor (Tgfbr1) in the periderm display a complete cleft of the secondary palate. Our subsequent experiments demonstrated that Tgfbr1-deficient periderm fails to undergo appropriate dedifferentiation. These studies define the periderm cell fate during palatogenesis and reveal a novel, critical role for TGF-β signaling in periderm dedifferentiation, which is a prerequisite for appropriate palatal epithelial adhesion and fusion.

Aberrant glial activation and synaptic defects in CaMKIIα-iCre and nestin-Cre transgenic mouse models

Current scientific research is driven by the ability to manipulate gene expression by utilizing the Cre/loxP system in transgenic mouse models. However, artifacts in Cre-driver mouse lines that introduce undesired effects and confound results are increasingly being reported. Here, we show aberrant neuroinflammation and synaptic changes in two widely used Cre-driver mouse models. Neuroinflammation in CaMKIIα-iCre mice was characterized by the activation and proliferation of microglia and astrocytes in synaptic layers of the hippocampus. Increased GFAP and Iba1 levels were observed in hippocampal brain regions of 4-, 8- and 22-month-old CaMKIIα-iCre mice compared to WT littermates. Synaptic changes in NMDAR, AMPAR, PSD95 and phosphorylated CaMKIIα became apparent in 8-month-old CaMKIIα-iCre mice but were not observed in 4-month-old CaMKIIα-iCre mice. Synaptophysin and synaptoporin were unchanged in CaMKIIα-iCre compared to WT mice, suggesting that synaptic alterations may occur in excitatory postsynaptic regions in which iCre is predominantly expressed. Finally, hippocampal volume was reduced in 22-month-old CaMKIIα-iCre mice compared to WT mice. We tested the brains of mice of additional common Cre-driver mouse models for neuroinflammation; the nestin-Cre mouse model showed synaptic changes and astrocytosis marked by increased GFAP+ astrocytes in cortical and hippocampal regions, while the original CaMKIIα-Cre T29-1 strain was comparable to WT mice. The mechanisms underlying abnormal neuroinflammation in nestin-Cre and CaMKIIα-iCre are unknown but may be associated with high levels of Cre expression. Our findings are critical to the scientific community and demonstrate that the correct Cre-driver controls must be included in all studies using these mice.

Generation of Pmel-dependent conditional and inducible Cre-driver mouse line for melanocytic-targeted gene manipulation.

Conditional and inducible gene targeting using Cre/loxP-mediated recombination is a powerful reverse genetics approach used to study spatiotemporal gene functions in specified cell types. To enable temporal gene manipulation in the melanocyte lineage, we established a novel inducible Cre-driver mouse line by targeting an all-in-one tetracycline/doxycycline (Dox)-inducible Cre expression cassette into the Pmel locus (PmelP2A-TetON3G-TRE3G-iCre ), a gene locus preferentially expressed in pigment cells. By crossing these Cre-driver mice with a strong Cre-reporter mouse line, Gt(ROSA)26Sortm9(CAG-tdTomato)Hze , we show the effectiveness of the PmelP2A-TetON3G-TRE3G-iCre mouse line in facilitating Dox-inducible Cre/loxP recombination in a wide variety of pigment cell lineages including hair follicle melanocytes and their stem cells. Furthermore, to demonstrate proof of concept, we ablated Notch signaling postnatally in the PmelP2A-TetON3G-TRE3G-iCre mice. In agreement with the previously reported phenotype, induced ablation of Notch signaling in the melanocyte lineage resulted in premature hair graying, demonstrating the utility of the PmelP2A-TetON3G-TRE3G-iCre allele. Therefore, the PmelP2A-TetON3G-TRE3G-iCre mouse line is suitable for assessing gene functions in melanocytes using an in vivo inducible reverse genetics approach. Furthermore, we unexpectedly identified previously unrecognized PMEL-expressing cells in non-pigmentary organs in the mice, suggesting unanticipated functions of PMEL other than melanosome formation.

Selective optogenetic activation of NaV1.7-expressing afferents in NaV1.7-ChR2 mice induces nocifensive behavior without affecting responses to mechanical and thermal stimuli.

In small and large spinal dorsal root ganglion neurons, subtypes of voltage-gated sodium channels, such as NaV1.7, NaV1.8, and NaV1.9 are expressed with characteristically localized and may play different roles in pain transmission and intractable pain development. Selective stimulation of each specific subtype in vivo may elucidate its role of each subtype in pain. So far, this has been difficult with current technology. However, Optogenetics, a recently developed technique, has enabled selective activation or inhibition of specific neural circulation in vivo. Moreover, optogenetics had even been used to selectively excite NaV1.8-expressing dorsal root ganglion neurons to induce nocifensive behavior. In recent years, genetic modification technologies such as CRISPR/Cas9 have advanced, and various knock-in mice can be easily generated using such technology. We aimed to investigate the effects of selective optogenetic activation of NaV1.7-expressing afferents on mouse behavior. We used CRISPR/Cas9-mediated homologous recombination to generate bicistronic NaV1.7-iCre knock-in mice, which express iCre recombinase under the endogenous NaV1.7 gene promoter without disrupting NaV1.7. The Cre-driver mice were crossed with channelrhodopsin-2 (ChR2) Cre-reporter Ai32 mice to obtain NaV1.7iCre/+;Ai32/+, NaV1.7iCre/iCre;Ai32/+, NaV1.7iCre/+;Ai32/Ai32, and NaV1.7iCre/iCre;Ai32/Ai32 mice. Compared with wild-type mice behavior, no differences were observed in the behaviors associated with mechanical and thermal stimuli exhibited by mice of the aforementioned genotypes, indicating that the endogenous NaV1.7 gene was not affected by the targeted insertion of iCre. Blue light irradiation to the hind paw induced paw withdrawal by mice of all genotypes in a light power-dependent manner. The threshold and incidence of paw withdrawal and aversive behavior in a blue-lit room were dependent on ChR2 expression level; the strongest response was observed in NaV1.7iCre/iCre;Ai32/Ai32 mice. Thus, we developed a non-invasive pain model in which peripheral nociceptors were optically activated in free-moving transgenic NaV1.7-ChR2 mice.

Distinct local and brain-wide networks are activated by optogenetic stimulation of neurons specific to each layer of motor cortex

Primary motor cortex (M1) consists of a stack of interconnected but distinct layers (L1-L6) which affect motor control through large-scale networks. However, the brain-wide functional influence of each layer is poorly understood. We sought to expand our knowledge of these layers’ circuitry by combining Cre-driver mouse lines, optogenetics, fMRI, and electrophysiology. Neuronal activities initiated in Drd3 neurons (within L2/3) were mainly confined within M1, while stimulation of Scnn1a, Rbp4, and Ntsr1 neurons (within L4, L5, and L6, respectively) evoked distinct responses in M1 and motor-related subcortical regions, including striatum and motor thalamus. We also found that fMRI responses from targeted stimulations correlated with both local field potentials (LFPs) and spike changes. This study represents a step forward in our understanding of how different layers of primary motor cortex are embedded in brain-wide circuitry.

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.

PHACTR-1 (Phosphatase and Actin Regulator 1) Deficiency in Either Endothelial or Smooth Muscle Cells Does Not Predispose Mice to Nonatherosclerotic Arteriopathies in 3 Transgenic Mice

Genome-wide association studies have revealed robust associations of common genetic polymorphisms in an intron of the PHACTR-1 (phosphatase and actin regulator 1) gene (chr6p24), with cervical artery dissection, spontaneous coronary artery dissection, and fibromuscular dysplasia. The aim was to assess its role in the pathogenesis of cervical artery dissection or fibromuscular dysplasia. Using various tissue-specific Cre-driver mouse lines, Phactr1 was deleted either in endothelial cells using 2 tissue-specific Cre-driver (PDGFB [platelet-derived growth factor B]-CreERT2 mice and Tie2 [tyrosine kinase with immunoglobulin and EGF homology domains]-Cre) and smooth muscle cells (smooth muscle actin-CreERT2) with a third tissue-specific Cre-driver. To test the efficacy of the Phactr1 deletion after cre-induction, we confirmed first, a decrease in Phactr1 transcription and Phactr1 expression in endothelial cell and smooth muscle cell isolated from Phactr1iPDGFB and Phactr1iSMA mice. Irrespective to the tissue or the duration of the deletion, mice did not spontaneously display pathological phenotype or vascular impairment: mouse survival, growth, blood pressure, large vessel morphology, or actin organization were not different in knockout mice than their comparatives littermates. Challenging vascular function and repair either by angiotensin II-induced hypertension or limb ischemia did not lead to vascular morphology or function impairment in Phactr1-deleted mice. Similarly, there were no more consequences of Phactr1 deletion during embryogenesis in endothelial cells. Loss of PHACTR-1 function in the cells involved in vascular physiology does not appear to induce a pathological vascular phenotype. The in vivo effect of the intronic variation described in genome-wide association studies is unlikely to involve downregulation in PHACTR-1 expression.