Knock-in Allele Research Articles

Phosphatidylserine phospholipase A1 enables GPR34-dependent immune cell accumulation in the peritoneal cavity.

The peritoneal cavity (PerC) is an important site for immune responses to infection and cancer metastasis. Yet few ligand-receptor axes are known to preferentially govern immune cell accumulation in this compartment. GPR34 is a lysophosphatidylserine (lysoPS)-responsive receptor that frequently harbors gain-of-function mutations in mucosa-associated B cell lymphoma. Here, we set out to test the impact of a GPR34 knock-in (KI) allele in the B-lineage. We report that GPR34 KI promotes the PerC accumulation of plasma cells (PC) and memory B cells (MemB). These KI cells migrate robustly to lysoPS ex vivo, and the KI allele synergizes with a Bcl2 transgene to promote MemB but not PC accumulation. Gene expression and labeling studies reveal that GPR34 KI enhances PerC MemB proliferation. Both KI PC and MemB are specifically enriched at the omentum, a visceral adipose tissue containing fibroblasts that express the lysoPS-generating PLA1A enzyme. Adoptive transfer and chimera experiments revealed that KI PC and MemB maintenance in the PerC is dependent on stromal PLA1A. These findings provide in vivo evidence that PLA1A produces lysoPS that can regulate GPR34-mediated immune cell accumulation at the omentum.

The embryonic zebrafish brain is exclusively colonized by pu.1-dependent and lymphatic-independent population of microglia.

Microglia, the crucial immune cells inhabiting the central nervous system (CNS), perform a range of vital functions, encompassing immune defense and neuronal regulation. Microglia subsets with diverse functions and distinct developmental regulations have been identified recently. It is generally accepted that all microglia originate from hematopoiesis and depend on the myeloid transcription factor PU.1. However, a recent study reported the existence of mrc1+ microglia in zebrafish embryos, which are seemingly independent of Pu.1 and reliant on lymphatic vessels, sparking great interest in the possibility of lymphatic-originated microglia. To address this, we took advantage of a pu.1 knock-in zebrafish allele for a detailed investigation. Our results conclusively showed that almost all zebrafish embryonic microglia (~95% on average) express pu.1. Further, lineage tracing and mutant analysis revealed that these microglia neither emerged from nor depended on lymphatic vessels. In essence, our study refutes the presence of pu.1-independent but lymphatic-dependent microglia.

Cortical neurodegeneration caused by Psen1 mutations is independent of Aβ

Mutations in the PSEN genes are the major cause of familial Alzheimer's disease, and presenilin (PS) is the catalytic subunit of γ-secretase, which cleaves type I transmembrane proteins, including the amyloid precursor protein (APP) to release Aβ peptides. While PS plays an essential role in the protection of neuronal survival, PSEN mutations also increase the ratio of Aβ42/Aβ40. Thus, it remains unresolved whether PSEN mutations cause AD via a loss of its essential function or increases of Aβ42/Aβ40. Here, we test whether the knockin (KI) allele of Psen1 L435F, the most severe FAD mutation located closest to the active site of γ-secretase, causes age-dependent cortical neurodegeneration independent of Aβ by crossing various Psen mutant mice to the App-null background. We report that removing Aβ completely through APP deficiency has no impact on the age-dependent neurodegeneration in Psen mutant mice, as shown by the absence of effects on the reduced cortical volume and decreases of cortical neurons at the ages of 12 and 18 mo. The L435F KI allele increases Aβ42/Aβ40 in the cerebral cortex while decreasing de novo production and steady-state levels of Aβ42 and Aβ40 in the presence of APP. Furthermore, APP deficiency does not alleviate elevated apoptotic cell death in the cerebral cortex of Psen mutant mice at the ages of 2, 12, and 18 mo, nor does it affect the progressive microgliosis in these mice. Our findings demonstrate that Psen1 mutations cause age-dependent neurodegeneration independent of Aβ, providing further support for a loss-of-function pathogenic mechanism underlying PSEN mutations.

Defining spatial nonuniformities of all ipRGC types using an improved Opn4cre recombinase mouse line

Intrinsically photosensitive retinal ganglion cells (ipRGCs) play a crucial role in several physiological light responses. In this study, we generate an improved Opn4cre knockin allele (Opn4cre(DSO)), which faithfully reproduces endogenous Opn4 expression and improves compatibility with widely used reporters. We evaluated the efficacy and sensitivity of Opn4cre(DSO) for labeling in retina and brain and provide an in-depth comparison with the extensively utilized Opn4cre(Saha) line. Through this characterization, Opn4cre(DSO) demonstrated higher specificity in labeling ipRGCs with minimal recombination escape. Leveraging a combination of electrophysiological, molecular, and morphological analyses, we confirmed its sensitivity in detecting all ipRGC types (M1-M6) and defined their unique topographical distribution across the retina. In the brain, the Opn4cre(DSO) line labels ipRGC projections with minimal labeling of cell bodies. Overall, the Opn4cre(DSO) mouse line represents an improved tool for studying ipRGC function and distribution, offering a means to selectively target these cells to study light-regulated behaviors and physiology.

Sex-Specific Response to A1BG Loss Results in Female Dilated Cardiomyopathy.

Cardiac disease often manifests differently in terms of frequency and pathology between men and women. However, the mechanisms underlying these differences are not fully understood. The glycoprotein A1BG is necessary for proper cardiac function in females but not males. Despite this, the role of A1BG in the female heart remains poorly studied. To determine the sex differential function of A1BG, we generated a novel conditional A1bg allele and a novel conditional A1bg Rosa26 knockin allele. Histology, electrocardiography, transcriptional profiling (RNA-seq), transmission electron microscopy, western blot analyses, mass spectrometry, and immunohistochemistry were used to assess cardiac structure and function. The study reveals that the absence of A1BG results in significant cardiac dysfunction in female but not male mice. Gene expression underscores that A1BG plays a critical role in metabolic processes and the integrity of intercalated discs in female cardiomyocytes. This dysfunction may be related to sex-specific A1BG cardiac interactomes and manifests as structural and functional alterations in the left ventricle indicative of dilated cardiomyopathy, thus suggesting a sex-specific requirement for A1BG in cardiac health. The loss of A1BG in cardiomyocytes leads to dilated cardiomyopathy in females, not males.

Comparative analysis of two independent Myh6-Cre transgenic mouse lines

We have previously shown that the Myh6 promoter drives Cre expression in a subset of male germ line cells in three independent Myh6-Cre mouse lines, including two transgenic lines and one knock-in allele. In this study, we further compared the tissue-specificity of the two Myh6-Cre transgenic mouse lines, MDS Myh6-Cre and AUTR Myh6-Cre, through examining the expression of tdTomato (tdTom) red fluorescence protein in multiple internal organs, including the heart, brain, liver, lung, pancreas and brown adipose tissue. Our results show that MDS Myh6-Cre mainly activates tdTom reporter in the heart, whereas AUTR Myh6-Cre activates tdTom expression significantly in the heart, and in the cells of liver, pancreas and brain. In the heart, similar to MDS Myh6-Cre,AUTR Myh6-Cre activates tdTom in most cardiomyocytes. In the other organs, AUTR Myh6-Cre not only mosaically activates tdTom in some parenchymal cells, such as hepatocytes in the liver and neurons in the brain, but also turns on tdTom in some interstitial cells of unknown identity.

Ectopic Activation of Fgf8 in Dental Mesenchyme Causes Incisor Agenesis and Molar Microdontia.

Putatively, tooth agenesis was attributed to the initiation failure of tooth germs, though little is known about the histological and molecular alterations. To address if constitutively active FGF signaling is associated with tooth agenesis, we activated Fgf8 in dental mesenchyme with Osr-cre knock-in allele in mice (Osr2-creKI; Rosa26R-Fgf8) and found incisor agenesis and molar microdontia. The cell survival assay showed tremendous apoptosis in both the Osr2-creKI; Rosa26R-Fgf8 incisor epithelium and mesenchyme, which initiated incisor regression from cap stage. In situ hybridization displayed vanished Shh transcription, and immunostaining exhibited reduced Runx2 expression and enlarged mesenchymal Lef1 domain in Osr2-creKI; Rosa26R-Fgf8 incisors, both of which were suggested to enhance apoptosis. In contrast, Osr2-creKI; Rosa26R-Fgf8 molar germs displayed mildly suppressed Shh transcription, and the increased expression of Ectodin, Runx2 and Lef1. Although mildly smaller than WT controls prenatally, the Osr2-creKI; Rosa26R-Fgf8 molar germs produced a miniature tooth with impaired mineralization after a 6-week sub-renal culture. Intriguingly, the implanted Osr2-creKI; Rosa26R-Fgf8 molar germs exhibited delayed odontoblast differentiation and accelerated ameloblast maturation. Collectively, the ectopically activated Fgf8 in dental mesenchyme caused incisor agenesis by triggering incisor regression and postnatal molar microdontia. Our findings reported tooth agenesis resulting from the regression from the early bell stage and implicated a correlation between tooth agenesis and microdontia.

TrkA + sensory neurons regulate osteosarcoma proliferation and vascularization to promote disease progression.

Bone pain is a presenting feature of bone cancers such as osteosarcoma (OS), relayed by skeletal-innervating peripheral afferent neurons. Potential functions of tumor-associated sensory neurons in bone cancers beyond pain sensation are unknown. To uncover neural regulatory functions, a chemical-genetic approach in mice with a knock-in allele for TrkA was used to functionally perturb sensory nerve innervation during OS growth and disease progression. TrkA inhibition in transgenic mice led to significant reductions in sarcoma-associated sensory innervation and vascularization, tumor growth and metastasis, and prolonged overall survival. Single-cell transcriptomics revealed that sarcoma denervation was associated with phenotypic alterations in both OS tumor cells and cells within the tumor microenvironment, and with reduced calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF) signaling. Multimodal and multi-omics analyses of human OS bone samples and human dorsal root ganglia neurons further implicated peripheral innervation and neurotrophin signaling in OS tumor biology. In order to curb tumor-associated axonal ingrowth, we next leveraged FDA-approved bupivacaine liposomes leading to significant reductions in sarcoma growth, vascularity, as well as alleviation of pain. In sum, TrkA-expressing peripheral neurons positively regulate key aspects of OS progression and sensory neural inhibition appears to disrupt calcitonin receptor signaling (CALCR) and VEGF signaling within the sarcoma microenvironment leading to significantly reduced tumor growth and improved survival. These data suggest that interventions to prevent pathological innervation of osteosarcoma represent a novel adjunctive therapy to improve clinical outcomes and survival.

Striatal GDNF Neurons Chemoattract RET-Positive Dopamine Axons at Seven Times Farther Distance Than Medium Spiny Neurons.

Glial cell line-derived neurotrophic factor (GDNF) is among the strongest dopamine neuron function- and survival-promoting factors known. Due to this reason, it has clinical relevance in dopamine disorders such as Parkinson's disease and schizophrenia. In the striatum, GDNF is exclusively expressed in interneurons, which make up only about 0.6% of striatal cells. Despite clinical significance, histological analysis of striatal GDNF system arborization and relevance to incoming dopamine axons, which bear its receptor RET, has remained enigmatic. This is mainly due to the lack of antibodies able to visualize GDNF- and RET-positive cellular processes; here, we overcome this problem by using knock-in marker alleles. We find that GDNF neurons chemoattract RET+ axons at least seven times farther in distance than medium spiny neurons (MSNs), which make up 95% of striatal neurons. Furthermore, we provide evidence that tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, is enriched towards GDNF neurons in the dopamine axons. Finally, we find that GDNF neuron arborizations occupy approximately only twelve times less striatal volume than 135 times more abundant MSNs. Collectively, our results improve our understanding of how endogenous GDNF affects striatal dopamine system function.

Valine and Inflammation Drive Epilepsy in a Mouse Model of ECHS1 Deficiency.

ECHS1 Deficiency (ECHS1D) is a rare and devastating pediatric disease that currently has no defined treatments. This disorder results from missense loss-of-function mutations in the ECHS1 gene that result in severe developmental delays, encephalopathy, hypotonia, and early death. ECHS1 enzymatic activity is necessary for the beta-oxidation of fatty acids and the oxidation of branched-chain amino acids within the inner mitochondrial matrix. The pathogenesis of disease remains unknown, however it is hypothesized that disease is driven by an accumulation of toxic metabolites from impaired valine oxidation. To expand our knowledge on disease mechanisms, a novel mouse model of ECHS1D was generated that possesses a disease-associated knock-in (KI) allele and a knock-out (KO) allele. To investigate the behavioral phenotype, a battery of testing was performed at multiple time points, which included assessments of learning, motor function, endurance, sensory responses, and anxiety. Neurological abnormalities were assessed using wireless telemetry EEG recordings, pentylenetetrazol (PTZ) seizure induction, and immunohistochemistry. Metabolic perturbations were measured within the liver, serum, and brain using mass spectrometry and magnetic resonance spectroscopy. To test disease mechanisms, mice were subjected to disease pathway stressors and then survival, body weight gain, and epilepsy were assessed. Mice containing KI/KI or KI/KO alleles were viable with normal development and survival, and the presence of KI and KO alleles resulted in a significant reduction in ECHS1 protein. ECHS1D mice displayed reduced exercise capacity and pain sensation. EEG analysis revealed increased slow wave power that was associated with perturbations in sleep. ECHS1D mice had significantly increased epileptiform EEG discharges, and were sensitive to seizure induction, which resulted in death of 60% of ECHS1D mice. Under basal conditions, brain structure was grossly normal, although histological analysis revealed increased microglial activation in aged ECHS1D mice. Increased dietary valine only affected ECHS1D mice, which significantly exacerbated seizure susceptibility and resulted in death. Lastly, acute inflammatory challenge drove regression and early lethality in ECHS1D mice. In conclusion, we developed a novel model of ECHS1D that may be used to further knowledge on disease mechanisms and to develop therapeutics. Our data suggests altered metabolic signaling and inflammation may contribute to epilepsy in ECHS1D, and these alterations may be attributed to impaired valine metabolism.

A knock-in allele of Hand2 expressing Dre recombinase.

HAND2 is a basic helix-loop-helix transcription factor with diverse functions during development. To facilitate the investigation of genetic and functional diversity among Hand2-expressing cells in the mouse, we have generated Hand2Dre, a knock-in allele expressing Dre recombinase. To avoid disrupting Hand2 function, the Dre cDNA is inserted at the 3' end of the Hand2 coding sequence following a viral 2A peptide. Hand2Dre homozygotes can therefore be used in complex crosses to increase the proportion of useful genotypes among offspring. Dre expression in mid-gestation Hand2Dre embryos is indistinguishable from wild-type Hand2 expression, and HandDre efficiently recombines rox target sites in vivo. In combination with existing Cre and Flp mouse lines, Hand2Dre will therefore extend the ability to perform genetic intersectional labeling, fate mapping, and functional manipulation of subpopulations of cells characterized by developmental expression of Hand2.

BDNF/TrkB Induces pCREBS133 Phosphorylation and Activates Mitochondrial Biogenesis in Rat Phrenic Motor Neurons

Neural control of the diaphragm muscle (DIAm) is essential to sustain breathing. During breathing, fatigue resistant DIAm motor units are recruited comprising smaller phrenic motor neurons (PhMNs) and type I and IIa muscle fibers. Larger PhMNs comprise more fatigable DIAm motor units that are infrequently active during expulsive behaviors. The difference in activation history is reflected in the higher mitochondrial volume density in smaller PhMNs. In PhMNs, brain-derived neurotrophic factor (BDNF) signals through its high-affnity receptor, tropomyosin receptor kinase B (TrkB.FL). Previously, we found that intrathecal BDNF treatment induces CREB phosphorylation at Serine 133 (pCREBs133). We hypothesized that pCREBs133 binds to the PGC1a promoter, transcriptionally targeting PGC1a expression, mediating downstream increased expression of NRF1, NRF2, and TFAM and mitochondrial biogenesis. To test this hypothesis, we used a chemosensitive TrkBF616 rat model with a knock-in allele sensitive to 1NMPP1 in that TrkB kinase is inhibited following 1NMPP1, a small molecule derivative of the protein kinase inhibitor protein phosphatase 1 that plays a significant role inhibiting the TrkB phosphorylation and activation. Systemic treatment of 1NMPP1 in TrkBF616 rats inhibits TrkB kinase activity. A bioinformatic analysis was performed to identify a putative binding site for pCREBs133 in the PGC1a promoter sequence in rats. Using a chromatin immunoprecipitation (ChIP) assay we found that pCREBS133 binds to the PGC1a promoter in rat cervical ventral horn. We showed that intrathecal BDNF treatment increased pCREBs133 phosphorylation, which was blocked by 1NMPP1 treatment in TrkBF616 rats. PGC1a, NRF1/2, and TFAM protein was compared in cervical ventral horn lysates from BDNF- and BDNF+1NMPP1-treated TrkBF616 rats by qRT-PCR and Western Blot. Relative mtDNA copy number (normalized to nuclear DNA) was quantified by qPCR to assess the mitochondrial biogenesis. All results were analyzed by paired t-test. Following 1NMPP1-induced inhibition of TrkB kinase, PGC1a, NRF1/2, and TFAM mRNA and protein expression was reduced. Consistent with these findings, mtDNA copy number was decreased following TrkB kinase inhibition by 1NMPP1. We conclude that inhibition of TrkB kinase by 1NMPP1 reduces PGC1a-mediated mitochondrial biogenesis in rat cervical ventral horn. Supported by NIH grants AG44615 and HL146114. 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.

Lack of effect from genetic deletion of Hdac6 in a humanized mouse model of CMT2D.

Inhibition of HDAC6 has been proposed as a broadly applicable therapeutic strategy for Charcot-Marie-Tooth disease (CMT). Inhibition of HDAC6 increases the acetylation of proteins important in axonal trafficking, such as α-tubulin and Miro, and has been shown to be efficacious in several preclinical studies using mouse models of CMT. Here, we sought to expand on previous preclinical studies by testing the effect of genetic deletion of Hdac6 on mice carrying a humanized knockin allele of Gars1, a model of CMT-type 2D. Gars1ΔETAQ mice were bred to an Hdac6 knockout strain, and the resulting offspring were evaluated for clinically relevant outcomes. The genetic deletion of Hdac6 increased α-tubulin acetylation in the sciatic nerves of both wild-type and Gars1ΔETAQ mice. However, when tested at 5 weeks of age, the Gars1ΔETAQ mice lacking Hdac6 showed no changes in body weight, muscle atrophy, grip strength or endurance, sciatic motor nerve conduction velocity, compound muscle action potential amplitude, or peripheral nerve histopathology compared to Gars1ΔETAQ mice with intact Hdac6. Our results differ from those of two previous studies that demonstrated the benefit of the HDAC6 inhibitor tubastatin A in mouse models of CMT2D. While we cannot fully explain the different outcomes, our results offer a counterexample to the benefit of inhibiting HDAC6 in CMT2D, suggesting additional research is necessary.

Abstract 3938: A novel mouse model reveals potential therapeutic strategies for RIT1-driven lung adenocarcinomas

Abstract Gain-of-function oncogenic mutations in RIT1 have been identified in lung adenocarcinoma and are mutually exclusive with other reported driver mutations, highlighting the role of RIT1 as a cancer driver. RIT1 is a poorly-studied non-classical RAS GTPase and, to date, there are no reported genetically-engineered mouse models of RIT1-driven lung adenocarcinoma. Here, we have generated a conditional knock-in allele within the endogenous mouse Rit1 locus, introducing the most frequent mutation observed in lung adenocarcinomas (RIT1M90I). Upon intranasal administration of adenovirus encoding the Cre recombinase to induce somatic recombination in the mouse lungs, we observed the formation of discrete adenomas. Although Rit1M90I mutation can promote lung tumor initiation in mice, we found that this phenotype is less penetrant than the common KrasG12D mutation. Immunohistochemistry analysis of Rit1M90I and KrasG12D tumors showed expression of typical histological markers associated with lung adenocarcinoma (i.e., TTF1 and SPC), as well as increased levels of Ki67 and pERK expression. Next, to develop potential pharmacological strategies for RIT1-driven lung tumors, we first explored drugs that inhibit the RAS/MAPK pathway and are either FDA-approved or under clinical development. In vitro experiments with cell lines carrying the RIT1M90I mutation demonstrated high sensitivity to SHP2, MEK, RAF, and RAS inhibitors. In vivo, SHP2 and RAS, but not MEK, inhibitors slowed down tumor progression in both cell line and patient-derived xenografts. Our results indicate that, although RAS signaling might be needed for RIT1-dependent growth, these tumors are not sensitive to MAPK inhibition as a monotherapy and, hence, other pathways could contribute to tumor growth. Therefore, we decided to explore whether targeting RIT1 directly, could be a more beneficial strategy for these tumors. In order to inhibit oncogenic RIT1 directly, we have undertaken two complementary approaches. First, because RIT1 mutations were shown to impair its proteolysis, we explored whether restoring RIT1 proteasomal degradation could be a promising strategy. Recently, the development of Proteolysis Targeting Chimeras (PROTAC) has demonstrated that targeted degradation can be used as a pharmacological approach for the treatment of cancer. Therefore, we have tested successfully several proof-of-concept PROTAC approaches that caused degradation of oncogenic RIT1 in different cellular models. Second, we used a chemical biology approach to assess whether covalent inhibitors directed against the closely-related GTPase KRAS could cross-react and bind to oncogenic RIT1. We have identified a molecule that binds RIT1 and we are currently characterizing it in our preclinical models. Overall, our work provides comprehensive insights into the oncogenic role of RIT1 in lung cancer emphasizing its potential as a therapeutic target. Citation Format: Alessandro Mauro Mozzarelli, Antonio Cuevas-Navarro, Anatoly Urisman, Pau Castel. A novel mouse model reveals potential therapeutic strategies for RIT1-driven lung adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3938.

De novo variants in FRYL are associated with developmental delay, intellectual disability, and dysmorphic features

FRY-like transcription coactivator (FRYL) belongs to a Furry protein family that is evolutionarily conserved from yeast to humans. The functions of FRYL in mammals are largely unknown, and variants in FRYL have not previously been associated with a Mendelian disease. Here, we report fourteen individuals with heterozygous variants in FRYL who present with developmental delay, intellectual disability, dysmorphic features, and other congenital anomalies in multiple systems. The variants are confirmed de novo in all individuals except one. Human genetic data suggest that FRYL is intolerant to loss of function (LoF). We find that the fly FRYL ortholog, furry (fry), is expressed in multiple tissues, including the central nervous system where it is present in neurons but not in glia. Homozygous fry LoF mutation is lethal at various developmental stages, and loss of fry in mutant clones causes defects in wings and compound eyes. We next modeled four out of the five missense variants found in affected individuals using fry knockin alleles. One variant behaves as a severe LoF variant, whereas two others behave as partial LoF variants. One variant does not cause any observable defect in flies, and the corresponding human variant is not confirmed to be de novo, suggesting that this is a variant of uncertain significance. In summary, our findings support that fry is required for proper development in flies and that the LoF variants in FRYL cause a dominant disorder with developmental and neurological symptoms due to haploinsufficiency.

Long-read sequencing for fast and robust identification of correct genome-edited alleles: PCR-based and Cas9 capture methods.

Recent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, via homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles. Targeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis.

Creation of Knock-In Alleles of Insulin Receptor Tagged by Fluorescent Proteins mCherry or EYFP in Fruit Fly Drosophila melanogaster.

The insulin/insulin-like growth factor-like signaling (IIS) pathway is highly conserved across metazoans and regulates numerous physiological functions, including development, metabolism, fecundity, and lifespan. The insulin receptor (InR), a crucial membrane receptor in the IIS pathway, is known to be ubiquitously expressed in various tissues, albeit at generally low levels, and its subcellular localization remains incompletely characterized. In this study, we employed CRISPR-mediated mutagenesis in the fruit fly Drosophila to create knock-in alleles of InR tagged with fluorescent proteins (InR::mCherry or InR::EYFP). By inserting the coding sequence of the fluorescent proteins mCherry or EYFP near the end of the coding sequence of the endogenous InR gene, we could trace the natural InR protein through their fluorescence. As an example, we investigated epithelial cells of the male accessory gland (AG), an internal reproductive organ, and identified two distinct patterns of InR::mCherry localization. In young AG, InR::mCherry accumulated on the basal plasma membrane between cells, whereas in mature AG, it exhibited intracellular localization as multiple puncta, indicating endocytic recycling of InR during cell growth. In the AG senescence accelerated by the mutation of Diuretic hormone 31 (Dh31), the presence of InR::mCherry puncta was more pronounced compared to the wild type. These findings raise expectations for the utility of the newly created InR::mCherry/EYFP alleles for studying the precise expression levels and subcellular localization of InR. Furthermore, this fluorescently tagged allele approach can be extended to investigate other membrane receptors with low abundance, facilitating the direct examination of their true expression and localization.

Live imaging of Fibronectin 1a-mNeonGreen and Fibronectin 1b-mCherry knock-in alleles during early zebrafish development

Within the developing embryo, cells assemble and remodel their surrounding extracellular matrix during morphogenesis. Fibronectin is an extracellular matrix glycoprotein and is a ligand for several members of the Integrin adhesion receptor family. Here, we compare the expression pattern and loss of function phenotypes of the two zebrafish fibronectin paralogs fn1a and fn1b. We engineered two fluorescently tagged knock-in alleles to facilitate live in vivo imaging of the Fibronectin matrix. Genetic complementation experiments indicate that the knock-in alleles are fully functional. Fn1a-mNeonGreen and Fn1b-mCherry are co-localized in ECM fibers on the surface of the paraxial mesoderm and myotendinous junction. In 5-days old zebrafish larvae, Fn1a-mNeonGreen predominantly localizes to the branchial arches, heart ventricle, olfactory placode and within the otic capsule while Fn1b-mCherry is deposited at the pericardium, proximal convoluted tubule, posterior hindgut and at the ventral mesoderm/cardinal vein. We examined Fn1a-mNeonGreen and Fn1b-mCherry in maternal zygotic integrin α5 mutants and integrin β1a; β1b double mutants and find distinct requirements for these Integrins in assembling the two Fibronectins into ECM fibers in different tissues. Rescue experiments via mRNA injection indicate that the two fibronectins are not fully inter-changeable. Lastly, we examined cross-regulation between the two Fibronectins and find fn1a is necessary for normal Fn1b fibrillogenesis in the presomitic mesoderm, but fn1b is dispensable for the normal pattern of Fn1a deposition.

Mouse Gene-Targeting Strategies for Maximum Ease and Versatility.

Well-planned strategies are an essential prerequisite for any mutational analysis involving gene targeting. Consideration of the advantages or disadvantages of different methods will aid in the production of a final product that is both technically feasible and versatile. Strategies for gene-targeting experiments in the mouse are discussed, including the rationale behind some of the common elements of gene-targeting vectors, such as homologous DNA and the use of different site-specific recombinases. We detail positive and negative selection as well as screening strategies for homologous recombination events in embryonic stem (ES) cells. For the planning stages of making different types of alleles, we first consider general strategies and then provide details specific to either homologous recombination in ES cells or making alleles by gene editing with CRISPR-Cas in preimplantation embryos. The types of alleles considered are null or knockout alleles, reporter gene knock-in alleles, point mutations, and conditional null alleles.

Murine Ribonuclease 6 Limits Bacterial Dissemination during Experimental Urinary Tract Infection.

The ribonuclease (RNase) A superfamily encodes cationic antimicrobial proteins with potent microbicidal activity toward uropathogenic bacteria. Ribonuclease 6 (RNase6) is an evolutionarily conserved, leukocyte-derived antimicrobial peptide with potent microbicidal activity toward uropathogenic Escherichia coli (UPEC), the most common cause of bacterial urinary tract infections (UTIs). In this study, we generated Rnase6-deficient mice to investigate the hypothesis that endogenous RNase 6 limits host susceptibility to UTI. We generated a Rnase6EGFP knock-in allele to identify cellular sources of Rnase6 and determine the consequences of homozygous Rnase6 deletion on antimicrobial activity and UTI susceptibility. We identified monocytes and macrophages as the primary cellular sources of Rnase6 in bladders and kidneys of Rnase6EGFP/+ mice. Rnase6 deficiency (i.e., Rnase6EGFP/EGFP) resulted in increased upper urinary tract UPEC burden during experimental UTI, compared to Rnase6+/+ controls. UPEC displayed increased intracellular survival in Rnase6-deficient macrophages. Our findings establish that RNase6 prevents pyelonephritis by promoting intracellular UPEC killing in monocytes and macrophages and reinforce the overarching contributions of endogenous antimicrobial RNase A proteins to host UTI defense.