Cre Induction Research Articles

Endothelial Beta 1 Integrins are Necessary for Microvascular Function and Glucose Uptake.

Microvascular insulin delivery to myocytes is rate limiting for the onset of insulin-stimulated muscle glucose uptake. The structural integrity of capillaries of the microvasculature is regulated, in part, by a family of transmembrane adhesion receptors known as integrins, which are composed of an α and β subunit. The integrin β1 (itgb1) subunit is highly expressed in endothelial cells (EC). EC itgb1 is necessary for the formation of capillary networks during embryonic during development and its knockdown blunts the reactive hyperemia that manifests during ischemia reperfusion. We investigated the contribution of EC itgb1 in microcirculatory function and glucose uptake with emphasis in skeletal muscle. We hypothesized that loss of EC itgb1 would impair microvascular hemodynamics and glucose uptake during insulin stimulation, creating 'delivery'-mediated insulin resistance. An itgβ1 knockdown mouse model was developed to avoid lethality of embryonic gene knockout and the deteriorating health resulting from early post-natal inducible gene deletion. Mice with (itgb1fl/flSCLcre) and without (itgb1fl/fl) tamoxifen inducible stem cell leukemia cre recombinase (SLCcre) expression at 10 days post cre induction had comparable exercise tolerance and pulmonary and cardiac functions. Using robust in vivo experimental platforms (i.e., intravital microscopy and hyperinsulinemic-euglycemic clamp), we show that itgb1fl/flSCLcre mice compared to itgb1fl/fl littermates have, i) deficits in capillary flow rate, flow heterogeneity, and capillary density; ii) impaired insulin-stimulated glucose uptake despite sufficient transcapillary insulin efflux; and iii) reduced insulin-stimulated glucose uptake due to perfusion-limited glucose delivery. Thus, EC itgb1 is necessary for microcirculatory function and to meet the metabolic challenge of insulin stimulation.

Endothelial β1 Integrins are Necessary for Microvascular Function and Glucose Uptake.

Microvascular insulin delivery to myocytes is rate limiting for the onset of insulin-stimulated muscle glucose uptake. The structural integrity of capillaries of the microvasculature is regulated, in part, by a family of transmembrane adhesion receptors known as integrins, which are composed of an α and β subunit. The integrin β1 (itgβ1) subunit is highly expressed in endothelial cells (EC). EC itgβ1 is necessary for the formation of capillary networks during embryonic during development and its knockdown in adult mice blunts the reactive hyperemia that manifests during ischemia reperfusion. In this study we investigated the contribution of skeletal muscle EC itgβ1 in microcirculatory function and glucose uptake. We hypothesized that loss of EC itgβ1 would impair microvascular hemodynamics and glucose uptake during insulin stimulation, creating 'delivery'-mediated insulin resistance. An itgβ1 knockdown mouse model was developed to avoid lethality of embryonic gene knockout and the deteriorating health resulting from early post-natal inducible gene deletion. We found that mice with (itgβ1fl/flSCLcre) and without (itgβ1fl/fl) inducible stem cell leukemia cre recombinase (SLCcre) expression at 10 days post cre induction have comparable exercise tolerance and pulmonary and cardiac functions. We quantified microcirculatory hemodynamics using intravital microscopy and the ability of mice to respond to the high metabolic demands of insulin-stimulated muscle using a hyperinsulinemic-euglycemia clamp. We show that itgβ1fl/flSCLcre mice compared to itgβ1fl/fl littermates have, i) deficits in capillary flow rate, flow heterogeneity, and capillary density; ii) impaired insulin-stimulated glucose uptake despite sufficient transcapillary insulin efflux; and iii) reduced insulin-stimulated glucose uptake due to perfusion-limited glucose delivery. Thus, EC itgβ1 is necessary for microcirculatory function and to meet the metabolic challenge of insulin stimulation.

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.

Genetic targeting of lymphatic endothelial cells in mice: current strategies and future perspectives.

Lymphatic vessels within different organs have diverse developmental origins, depend on different growth factor signaling pathways for their development and maintenance, and display notable tissue-specific adaptations that contribute to their roles in normal physiology and in various diseases. Functional studies on the lymphatic vasculature rely extensively on the use of mouse models that allow selective gene targeting of lymphatic endothelial cells (LECs). Here, we discuss LEC diversity and provide an overview of some of the commonly used LEC-specific inducible Cre lines and induction protocols, outlining essential experimental parameters and their implications. We describe optimized treatment regimens for embryonic, postnatal and adult LECs, efficiently targeting organs that are commonly studied in lymphatic vascular research, such as the mesentery and skin. We further highlight the anticipated outcomes and limitations associated with each induction scheme and mouse line. The proposed protocols serve as recommendations for laboratories initiating studies involving targeting of the lymphatic vasculature, and aim to promote uniformity in lineage tracing and functional studies within the lymphatic vascular field.

Specificity and efficiency of tamoxifen-mediated Cre induction is equivalent regardless of age

Temporally controlling Cre recombination through tamoxifen (Tam) induction has many advantages for biomedical research. Most studies report early post-natal/juvenile (<2 m.o.) Tam induction, but age-related neurodegeneration and aging studies can require Cre induction in older mice (>12 m.o.). While anecdotally reported as problematic, there are no published comparisons of Tam-mediated Cre induction at early and late ages. Here, microglial-specific Cx3cr1creERT2 mice were crossed to a floxed NuTRAP reporter to compare Cre induction at early (3-6 m.o.) and late (20 m.o.) ages. Specificity and efficiency of microglial labeling at 21-22 m.o. were identical in mice induced with Tam at early and late ages. Age-related microglial translatomic changes were also similar regardless of Tam induction age. Each Cre and flox mouse line should be independently validated, however, these findings demonstrate that Tam-mediated Cre induction can be performed even into older mouse ages and should be generalizable to other inducible Cre models.

Lrig1+ small intestinal progenitor cells require MYO5B for proper crypt proliferation and enterocyte maturation

Microvillus inclusion disease (MVID) is a congenital disorder characterized by severe diarrhea. MVID is caused by inactivating mutations in myosin Vb (MYO5B), a motor protein. Mice lacking MYO5B in their intestinal epithelium have demonstrated the importance of MYO5B in enterocyte brush border formation and epithelial cell differentiation. Additionally, the proliferative zone of the intestinal crypt of MYO5B-deficient mice is elongated compared to controls. We hypothesize that the progenitor cell-specific Cre driver under Lrig1 demonstrates crypt-specific effects caused by MYO5B loss. Lrig1-CreERT2;R26R-YFP and Myo5bfl/fl mice were crossbred to generate Lrig1-CreERT2;R26R-YFP;MYO5B fl/fl (Lrig1 ΔMYO5B ) mice. Adult Lrig1 ΔMYO5B mice and control littermates received tamoxifen injection at day 0. GI tissues were collected 2.5, 3, 4, and 5 days after tamoxifen administration for histological characterization. Lrig1 ΔMYO5B mice began experiencing significant weight loss 4 days after tamoxifen injection and lost 20% of their starting body weight on day 5. At day 5, the Lrig1 ΔMYO5B mouse colon was devoid of solid feces and their small intestine contained clear luminal contents, together suggesting a watery diarrheal phenotype. Enterocytes of Lrig1DMYO5B mice on day 5 contained abnormal cytoplasmic accumulation of PAS staining. Fewer goblet cells were apparent in intestinal sections from day 3 and 5 Lrig1 ΔMYO5B mice compared to controls. Crypts were elongated starting at day 3. Similarly, the PCNA+ epithelial region was expanded in Lrig1 ΔMYO5B mice, even reaching into the villi at day 5. The time course of the PCNA+ region expansion corresponded to the wave of MYO5B-loss beginning at the base of crypts. MYO5B expression was diminished in the crypts at day 3 and nearly absent from the epithelium by day 5. Interestingly, expression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), the rate-limited enzyme of ketogenesis that is important for stem cell function, was expanded in the crypt at day 3 and 5 after tamoxifen. Sodium/glucose cotransporter 1 (SGLT1), which is present along the apical surface of mature enterocytes, was progressively diminished and mis-localized away from the apical surface after tamoxifen administration. Apical sodium-dependent bile acid transporter (ASBT), found in the distal small intestine, demonstrated a similar mis-localization away from the apical membrane in enterocytes.We established a novel mouse model that demonstrates progressive MYO5B loss in the intestinal epithelium, starting at the base of the crypt and reaching villi tips over the course of 5 days following Cre induction. This Lrig1 ΔMYO5B strain will allow for lineage tracing studies to better understand the effect of MYO5B loss on intestinal epithelial differentiation and progenitor function. NSF GFRP, NIH R01DK128190 This is the full abstract presented at the American Physiology Summit 2023 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.

DDB1 and CUL4 Associated Factor 7 (DCAF7) Is Essential for Hematopoiesis

Mutations in DDB1 And CUL4 Associated Factor 7 (DCAF7) have been detected in a subset of patients with Myeloid Leukemia in Down syndrome. DCAF7 is a highly conserved WD40 domain protein with 100% homology among vertebrates and is a prominent binding partner of DYRK1A, a kinase located on chromosome 21. Analysis of the DepMap database revealed that hematopoietic cell lines are highly dependent on DCAF7 compared to cell lines from other tumors. However, the function of DCAF7 during normal hematopoiesis is currently unknown. Thus, we aimed to study its function in hematopoietic cell lines and in a conditional knockout mouse model. We first deleted DCAF7 from a panel of hematopoietic cell lines by CRISPR/Cas9 editing and observed its absence led to cell cycle arrest and decreased proliferation. Rescue experiments in which a CRISPR resistant DCAF7 allele was expressed in the targeted clones restored proliferation and cell cycling. A comparison of RNA-seq data between parental HEL cells and those without DCAF7 showed decreased expression of genes involved in differentiation, TNF signaling and the IFN pathway, indicating that DCAF7 is important for cell differentiation. Next, we created a Dcaf7 conditionally targeted mouse strain (Dcaf7F/F Mx-1 Cre) and assessed its role in hematopoiesis in vivo. Following Cre induction by pIpC, we observed a decrease in white blood cells, hemoglobin and platelet counts in KO mice. Analysis of the bone marrow showed an accumulation of LT-HSCs and multipotent progenitors. In addition, we observed a defect in the generation of B-cells, reminiscent of the phenotype observed upon Dyr1ka deletion. We then tested the recovery of Dcaf7 KO hematopoietic cells after different types of stress, which included ex-vivo culture, transplantation, and myelosuppression. Dcaf7 deficient HSCs showed defective expansion in PVA-containing media and poor differentiation in single cell colony assays in methylcellulose. In non-competitive transplants, KO cells reconstituted hematopoiesis although the number of mature myeloid and lymphoid cells was dramatically decreased. In competitive transplants, Dcaf7 KO cells were outcompeted by wild-type cells. Lastly, upon myelosuppression with 5-FU, Dcaf7 deficient mice were not able to recover and died acutely. Overall, these results indicate that DCAF7 plays a critical role in HSCs during homeostasis and regeneration. To uncover the mechanisms by which DCAF7 contributes to hematopoiesis, we performed IP-MS using tagged DCAF7 as bait in a hematopoietic cell line. In addition to DYRK1A, we found that DCAF7 interacts with multiple components of the polycomb repressive complex1 (PRC1), specifically the non-canonical PRC1.5 complex, which has been previously implicated in gene activation and transcription. We confirmed that DCAF7 engages in the PRC1.5 complex by glycerol gradient centrifugation. Upon DCAF7 absence the size the PRC1 complex shifts towards a lower molecular weight, indicating that DCAF7 is a core subunit of the complex. We then performed RNASeq analysis in sorted LT-HSCs (Lin-cKit+Sca1+CD48-CD150+) and found that 70% of the differentially expressed genes were downregulated in KO cells. These downregulated genes were associated with diminished activation of GATA1, GATA2, RUNX1 and SP1 target genes, in agreement with the functional role of the PRC1.5 complex in gene activation and cell differentiation. Together our results reveal that DCAF7 regulates hematopoiesis by participating in transcriptional regulatory complexes, and we predict that DCAF7 mutations contribute to leukemia by cooperating with DYRK1A and altering the activity of the PRC1 complex.

Cdh5-lineage-independent origin of dermal lymphatics shown by temporally restricted lineage tracing.

The developmental origins of lymphatic endothelial cells (LECs) have been under intense research after a century-long debate. Although previously thought to be of solely venous endothelial origin, additional sources of LECs were recently identified in multiple tissues in mice. Here, we investigated the regional differences in the origin(s) of the dermal lymphatic vasculature by lineage tracing using the pan-endothelial Cdh5-CreER T2 line. Tamoxifen-induced labeling of blood ECs at E9.5, before initiation of lymphatic development, traced most of the dermal LECs but with lower efficiency in the lumbar compared with the cervical skin. By contrast, when used at E9.5 but not at E11.5, 4-hydroxytamoxifen, the active metabolite of tamoxifen that provides a tighter window of Cre activity, revealed low labeling frequency of LECs, and lymphvasculogenic clusters in the lumbar skin in particular. Temporally restricted lineage tracing thus reveals contribution of LECs of Cdh5-lineage-independent origin to dermal lymphatic vasculature. Our results further highlight Cre induction strategy as a critical parameter in defining the temporal window for stage-specific lineage tracing during early developmental stages of rapid tissue differentiation.

Machine Learning‐Guided Engineering of Cre‐lox Recombination for Comprehensive Analysis of Neural Networks

BackgroundNeural networks generate complex behaviors. However, the relationships between them have not been understood well. Thus, we have developed ‘functional cellomics’, which enables functional annotation of neural networks in a hypothesis‐free manner. The key is the stochastic labeling of opsin achieved by Cre‐mediated stochastic recombination. We designed a gene cassette, which contained two sets of lox variants, lox2272 and loxP, alternately. A transcription factor, QF2w, is interposed between loxP. Cre excises DNA with an exclusive choice of between lox2272 or loxP. When Cre excises lox2272, expressed QF2w induces opsin. Constructing a C. elegans strain carrying this gene cassette, we successfully implemented the stochastic labeling of opsin dependent on Cre induction [1]. However, the labeling rate of opsin was about 30 %. If a large subset of neurons is labeled by opsin, it becomes difficult to distinguish the function of each neuron. In this study, we demonstrated a strategy to precisely control the labeling rate of opsin by machine learning‐guided engineering of Cre‐lox recombination.MethodIn our genetic circuit, the opsin coding gene is expressed in neurons where Cre‐lox recombination occurs between lox2272 sequences. Therefore, we hypothesized that the labeling rate of opsin would be reduced by using lox2272 variants, which is difficult to be excised by Cre. Thus, we generated a library of randomized lox sequences by PCR and introduced them in Saccharomyces cerevisiae. We then induced Cre in the S. cerevisiae and extracted DNA from S. cerevisiae. We used NGS to assess the excision rate of the lox2272 variants. To develop a machine learning model to predict the excision rate of the lox2272 variants, we trained a Gaussian process (GP) model using data containing lox2272 sequences and their excision rates by Cre. Approximately 1000 lox2272 variants were randomly selected as training data and 100 variants were selected to test the prediction accuracy.Results and DiscussionWe quantified the excision rates of over 2000 lox2272 variants by NGS and successfully found lox2272 variants with excision efficiencies ranging from 0.05% to 100%. The results of the NGS analysis were confirmed by qPCR. Next, we created a GP model and observed a high correlation between the actual and predicted cleavage rates of the test data. Using the GP model, we predicted the efficiency of the unevaluated lox2272 sequence and successfully identified lox2272 variants that had various cleavage rates. These results demonstrate the feasibility of precise control of opsin labeling rates in functional cellomics [2].[1] Aoki et al, Sci Rep, 8, 10380, 2018[2] Yamauchi et al, submitted

Cx3Cr1-Cre induction leads to microglial activation and IFN-1 signaling caused by DNA damage in early postnatal brain.

Cx3cr1CreER-driven Cre recombinase (Cre) is a widely used genetic tool for enabling gene manipulation in microglia and macrophages. However, an in-depth analysis of the possible detrimental effects of Cre activity in microglia, surprisingly, remains missing. Here, we demonstrate an age-dependent sensitivity of microglia to Cx3cr1-Cre toxicity, wherein Cre induction, specifically in early postnatal microglia, is detrimental to microglial development, proliferation, and function. Tamoxifen (TAM)-induced Cre activity leads to microglial activation, type 1 interferon (IFN-1) signaling, and increased phagocytosis, causing aberrant synaptic pruning during the early postnatal period and anxious behavior at later age. The detrimental effects of Cre induction are caused by DNA-damage-induced toxicity in microglia and are limited to the early postnatal period, showing no detrimental effects in adult microglia. Thus, our study reveals an age-dependent vulnerability of microglia to Cre activity, thereby highlighting age dependency of Cre action, which could be especially applicable in the broader context of environment-responsive cell types.

A Conserved Extracellular Motif Regulates Signaling and Trafficking of ADGRG2

ADGRG2(GPR64) is an orphan adhesion G protein-coupled receptor (aGPCR), which is expressed in the epididymis and parathyroid glands. We have previously shown that ADGRG2 regulates the secretion of parathyroid hormone by parathyroid adenoma cells via interaction with and altering calcium-sensing receptor signaling. Generally, dissociation of a cleaved N-terminal Fragment (NTF) from the C-terminal fragment is a prerequisite for the activation of aGPCRs. Our previous data confirmed this mechanism in human ADGRG2, as well as the role of b-arrestins, dynamin, and GPCR kinases in the signaling of NTF-truncated ADGRG2. However, the structural motifs other than the NTF that regulate signaling and trafficking of ADGRG2 remain unknown. Herein, we mutated residues in the extracellular loop 2 (ECL2) that are conserved among zebrafish, mouse, and human ADGRG2, as well as the whole family of aGPCRs. Using HEK293 cells as a cell model, we elucidated how these mutations alter cAMP production and CRE induction (Biosensor and Luciferase Reporter Assays), cell resistance (label-free impedance assay), receptor surface expression (ELISA, Immunofluorescence imaging), and receptor degradation (Western blotting). We discovered that mutations in tryptophan and isoleucine of ECL2 significantly decrease surface and total expression of ADGRG2. Treatment with a translation inhibitor, cycloheximide, revealed that these mutants are degraded at a much higher rate compared to the WT receptor. Having adjusted the dose of plasmids to reach similar surface expression, we discovered that ECL2-mutated receptors do not respond to the activating synthetic peptide, P-15, in any of the signaling readouts. Using a fluorophore-conjugated P-15, we found that ECL2 plays a critical role in the binding of the activating peptide to ADGRG2. These data suggest a major role for ECL2 and its conserved residues in signaling, trafficking, and stability of ADGRG2.

A transgenic Alx4-CreER mouse to analyze anterior limb and nephric duct development.

Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited. We describe a transgenic mouse line expressing CreERT2 from the Aristaless-like 4 (Alx4) promoter that induces recombination in the anterior limb. Cre induction at embryonic day 8.5 revealed that Alx4-CreERT2 labeled cells using the mTmG Cre reporter contributed to anterior digits I to III as well as the radius of the forelimb. Cre activity is expanded further along the AP axis in the hindlimb than in the forelimb resulting in some Cre reporter cells contributing to digit IV. Induction at later time points labeled cells that become progressively restricted to more anterior digits and proximal structures. Comparison of Cre expression from the Alx4 promoter transgene with endogenous Alx4 expression reveals Cre expression is slightly expanded posteriorly relative to the endogenous Alx4 expression. Using Alx4-CreERT2 to induce loss of intraflagellar transport 88 (Ift88), a gene required for ciliogenesis, hedgehog signaling, and limb patterning, did not cause overt skeletal malformations. However, the efficiency of deletion, time needed for Ift88 protein turnover, and for cilia to regress may hinder using this approach to analyze cilia in the limb. Alx4-CreERT2 is also active in the mesonephros and nephric duct that contribute to the collecting tubules and ducts of the adult nephron. Embryonic activation of the Alx4-CreERT2 in the Ift88 conditional line results in cyst formation in the collecting tubules/ducts. Overall, the Alx4-CreERT2 line will be a new tool to assess cell fates and analyze gene function in the anterior limb, mesonephros, and nephric duct.

Optimization of tamoxifen-induced Cre activity and its effect on immune cell populations

Tamoxifen (TAM) inducible Cre recombinase system is an essential tool to study gene function when early ablation or overexpression can cause developmental defects or embryonic lethality. However, there remains a lack of consensus on the optimal route and dosage of TAM administration in vivo. Here, we assessed dosage and delivery of TAM for activation of Cre in immune cell subsets assessed longitudinally and spatially using transgenic mice with ubiquitously expressed Cre/ER and the Cre-inducible fluorescent reporter YFP. After comparing two TAM delivery methods (intraperitoneal versus oral gavage) and different doses, we found that 3 mg of TAM administered orally for five consecutive days provides maximal reporter induction with minimal adverse effects in vivo. Serum levels of TAM peaked 1 week after initiating treatment then slowly decreased, regardless of dosing and delivery methods. TAM concentration in specific tissues (liver, spleen, lymph nodes, and thymus) was also dependent on delivery method and dose. Cre induction was highest in myeloid cells and B cells and substantially lower in T cells, and double-positive thymocytes had a notably higher response to TAM. In addition to establishing optimal dose and administration of TAM, our study reveals a disparate activity of Cre in different cell immune populations when using Cre/ER models.

Abstract B12: Carcinogen exposure alters keratin 5 expression and K5-Cre recombination in transgenic mouse urothelium

Abstract In the urinary bladder, the normal urothelium is divided into three subpopulations of cells: superficial umbrella cells, intermediate cells, and a basal layer, each with distinct expression patterns of uroplakins (UP) and keratin 5 (K5). Many current transgenic mouse models (Tg) for urinary bladder cancer rely on uroplakin or keratin promoters to drive inducible Cre-lox recombination in basal or luminal bladder urothelial cells. Further, many models utilize the chemical carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), to drive or accelerate tumorigenesis. A major limitation in Tg model systems where genes are knocked out is the requirement for highly penetrant and reproducible Cre activity and subsequent gene deletion in order to see a phenotype. This problem is compounded in long-term experiments such as BBN carcinogenesis that can run for up to six months after Cre induction. We have previously shown that Trim29 (tripartite motif containing 29) is sufficient to drive invasive bladder cancer formation in Tg models and in human bladder tumors. We have now developed a Tg system utilizing K5-Cre-ERT2 to target Trim29 for deletion in K5-expressing basal urothelial cells. In this model, tamoxifen exposure results in Cre expression and Trim29 deletion in K5 basal cells. Whether this would translate into complete loss of Trim29 in the urothelium over time and what effect BBN might have on tamoxifen induction of K5-Cre and loss of Trim29 were unclear. We found that exposure of K5-Cre-ERT2, Trim29 flox/flox mice to tamoxifen for six weeks resulted in loss of Trim29 expression only in the K5+ basal urothelium and that these K5+ Trim29 -/- basal cells did not give rise to intermediate cells or umbrella cells during the six months of follow-up. In contrast, mice treated with 0.05% BBN in drinking water for six months after induction of Cre produced full thickness of Trim29 knockout (KO) in 50% of mice (8/16 mice). Our results indicate that in a nonstressed state, K5 basal cells give rise to other basal cells but do not repopulate intermediate or umbrella cell layers. Furthermore, exposure to BBN resulted in the proliferation of basal cells and the replacement of intermediate cells by basal precursors in the urothelium. These results suggest that K5-Cre promoters may have different effects on transgene recombination in the bladder in the presence or absence of BBN, which induces more universal expression of Cre and its subsequent recombination events. These findings should be considered when interpreting and developing BBN-induced bladder cancer Tg models. Citation Format: Alan Kelleher, Yin Wang, Luke Broses, Sylvia Zelenka-Wang, Phillip L. Palmbos. Carcinogen exposure alters keratin 5 expression and K5-Cre recombination in transgenic mouse urothelium [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B12.

Is skeletal muscle a functional immunosensory organ of pathogens? A murine model of polymicrobial sepsis.

The body’s ability to sense lipopolysaccharide (LPS) is critical in the inflammatory response to bacterial infection. LPS is a ligand for toll‐like receptors (TLRs), expressed on immune and many other cell types. When activated, they lead to secretion of cytokines such as IL‐6 and IL‐1ß. These cytokines, in turn, promote the activity and trafficking of inflammatory cells to the site of infection. We hypothesize that skeletal muscle (skm), which also expresses TLRs, plays a role in directing inflammatory cells via cytokine secretion during sepsis. In order to test this, we developed an inducible skeletal muscle‐specific knockdown of MYD88 (an adaptor protein necessary for TLR function) using the Mer‐CRE‐Mer system.METHODSMale and female mice on skmCRE background were injected with raloxifene to induce skmMYD88 knockdown vs. vehicle control (no CRE induction). Three weeks after CRE induction, sepsis was induced via intraperitoneal injection of a calibrated dose of cecal slurry. The animals were sacrificed 6 and 12 hours post‐injection. Plasma and peritoneal lavage were collected at the time of sacrifice, and analyzed for the presence of various inflammatory cells via a HESKA hematological analyzer.RESULTSskmMYD88−/− females showed a significant increase in the %neutrophils in the lavage at both the 6h (P&lt;0.05) and 12h time points (P&lt;0.0001) compared to skmMYD88+/+. The % of lavage basophils were elevated in female skmMYD88−/− at 12h (P&lt; 0.01), as were %eosinophils (P&lt;0.0001). Female lavage %monocytes increased at 12h in skmMYD88−/−. There were no differences in lavage %lymphocytes. No differences between the male skmMYD88−/− were observed for any cell types in the peritoneal lavage. In the blood, the only significant differences were found in males, in which the skmMYD88−/− group displayed increased %monocytes and decreased %lymphocytes at 12h.DISCUSSIONThe data suggest that skeletal muscle is capable of selectively governing the direction and presence of immune cells at the site infection, i.e. the peritoneum. The cause of this influence appears to be, in part, due to skeletal muscle TLR activation. While total leukocytes remain unchanged in the peritoneal lavage, knocking down skmMYD88 resulted an altered immune cell profile during the time‐course of infection. In the case of neutrophils, skeletal muscle appears to play a major suppressive regulatory role. Overall, these results implicate skeletal muscle as a significant player in pathogen recognition and response.Support or Funding InformationSupported by NIH 1R01GM118895 (TLC) and the BK Betty Stevens Endowment

High protein diet increases kidney macrophages and accelerates polycystic kidney disease

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is the most common genetic kidney disease that leads to end stage renal disease. Interestingly, the disease severity is highly variable even among families with the same gene mutation, suggesting factors other than genetics may affect cystogenesis. One factor that accelerates cystogenesis in PKD rodent is a high protein diet that results in renal hypertrophy. However, the mechanism of how a high protein diet promotes cystogenesis is unknown. We have shown that renal hypertrophy triggered by unilateral nephrectomy increases kidney macrophages and accelerates cystogenesis in ADPKD mouse. Therefore, we hypothesize that a high protein diet fed in ADPKD mice leads to increased kidney macrophages and accelerates cyst growth similar to a unilateral nephrectomy model.MethodsWe used adult tamoxifen inducible Pkd1flox/flox mice with or without Cagg cre‐ER. These adult conditional Pkd1 knockout mice typically requires months to develop major kidney cysts. Three weeks after cre induction, mice were fed with either a high protein (HP: 60%), a normal protein (NP: 18%) or a low protein (LP: 6%) diet for a total of 1 and 6 weeks. Some mice were given a HP diet and treated either with liposomal clodronate (to deplete macrophage) or with PBS (IP twice a week) for a total of 6 weeks. All mice were sacrificed at the end of the experiment and kidneys were harvested for histology, cytokines and flow cytometry for kidney immune cell sorting.ResultsPkd1 mice fed a HP diet for 6 weeks resulted in increased number of kidney resident macrophage (CD11blo, F4/80 hi) and infiltrating macrophages (CD11bhi, F4/80lo) compared to kidneys from a NP or LP diet fed mice. A HP diet resulted in increased kidney pro‐inflammatory cytokines, chemokine and developed severe kidney cysts compared to a NP or LP diet group. Pkd1 mice treated with liposomal clodronate had fewer resident and infiltrating macrophages and smaller kidneys compared to control PBS treated group. Interestingly, mice fed a HP diet for 1 week already had larger kidneys compared to those fed a LP diet, but there were no differences in kidney chemokines or tubular injury marker.ConclusionsRenal hypertrophy triggered by a HP diet increases kidney macrophages, inflammatory cytokine production and promotes cyst growth in adult Pkd1 mice. The mechanism of how dietary protein initiates and accelerates cyst growth remains to be determined.Support or Funding InformationNIH/NIDDK K08 DK106465, R03 DK119717 PKD Foundation 232G19a

One-step generation of zebrafish carrying a conditional knockout-knockin visible switch via CRISPR/Cas9-mediated intron targeting.

The zebrafish has become a popular vertebrate animal model in biomedical research. However, it is still challenging to make conditional gene knockout (CKO) models in zebrafish due to the low efficiency of homologous recombination (HR). Here we report an efficient non-HR-based method for generating zebrafish carrying a CKO and knockin (KI) switch (zCKOIS) coupled with dual-color fluorescent reporters. Using this strategy, we generated hey2zKOIS which served as a hey2 KI reporter with EGFP expression. Upon Cre induction in targeted cells, the hey2zCKOIS was switched to a non-functional CKO allele hey2zCKOIS-invassociated with TagRFP expression, enabling visualization of the CKO alleles. Thus, simplification of the design, and the visibility and combination of both CKO and KI alleles make our zCKOIS strategy an applicable CKO approach for zebrafish.

One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish.

CRISPR/Cas systems are widely used to knock out genes by inducing indel mutations, which are prone to genetic compensation. Complex genome modifications such as knockin (KI) might bypass compensation, though difficult to practice due to low efficiency. Moreover, no 'two-in-one' KI strategy combining conditional knockout (CKO) with fluorescent gene-labeling or further allele-labeling has been reported. Here, we developed a dual-cassette-donor strategy and achieved one-step and efficient generation of dual-function KI alleles at tbx5a and kctd10 loci in zebrafish via targeted insertion. These alleles display fluorescent gene-tagging and CKO effects before and after Cre induction, respectively. By introducing a second fluorescent reporter, geno-tagging effects were achieved at tbx5a and sox10 loci, exhibiting CKO coupled with fluorescent reporter switch upon Cre induction, enabling tracing of three distinct genotypes. We found that LiCl purification of gRNA is critical for highly efficient KI, and preselection of founders allows the efficient germline recovery of KI events.

Tamoxifen induction of Cre recombinase does not cause long-lasting or sexually divergent responses in the CNS epigenome or transcriptome: implications for the design of aging studies

The systemic delivery of tamoxifen (Tam) to activate inducible CreERT2-loxP transgenic mouse systems is now widely used in neuroscience studies. This critical technological advancement allows temporal control of DNA-cre recombination, avoidance of embryonically lethal phenotypes, and minimization of residual cell labeling encountered in constitutively active drivers. Despite its advantages, the use of Tam has the potential to cause long-lasting, uncharacterized side effects on the transcriptome and epigenome in the CNS, given its mixed estrogen receptor (ER) agonist/antagonist actions. With the welcome focus on including both sexes in biomedical studies and efforts to understand sex differences, Tam administration could also cause sexually divergent responses that would confound studies. To examine these issues, epigenetic and transcriptomic profiles were compared in C57BL/6J female and male hippocampus, cortex, and retina 1month after a 5-day Tam treatment typical for cre induction, or vehicle control (sunflower seed oil). Cytosine methylation and hydroxymethylation levels, in both CG and non-CG contexts, were unchanged as determined by oxidative bisulfite sequencing. Long-lasting Tam transcriptomic effects were also not evident/minimal. Furthermore, there is no evidence of sexually divergent responses with Tam administration and Tam did not alter sex differences evident in controls. Combined with recently reported data that Tam alone does not cause long-lasting changes in behavior and neurogenesis, our findings provide confidence that Tam can be used as a cre-recombinase inducer without introducing significant confounds in transcriptomic and epigenomic neuroscience studies, particularly those focused on genomic and transcriptomic aspects of the aging brain.

PS969 HYPOMETHYLATING DRUGS SPECIFICALLY TARGET DNMT3A MUTATION ASSOCIATED CLONAL HEMATOPOIESIS AND ACUTE MYELOID LEUKEMIA

Background:DNMT3A mutations commonly occur in myeloid malignancies and in clonal hematopoiesis. The codon arginine 882 (R882) is the major hotspot mutation in patients with de novo AML, in secondary AML as well as in individuals with clonal hematopoiesis in the absence of leukemic transformation. Although the majority of patients with DNMT3A‐R882H AML initially respond to conventional chemotherapy, many of them subsequently relapse, suggesting that DNMT3A‐R882H cells persist and drive relapse. No specific therapy exists to date for clonal hematopoiesis or DNMT3A‐R882H mutated AML. Recent studies have shown that DNMT inhibitors (DNMTi) could be particularly beneficial for patients with DNMT3A mutations. However, the extent of this association, the potential to cure patients and the mechanisms remain unresolved.Aims:Our aim is to identify in which context leukemic and pre‐leukemic stem cells with DNMT3A‐R882 mutation can be targeted by DNMT‐inhibitors as mono‐therapy and in combination.Methods:In order to understand the molecular and functional consequences of the DNMT3A‐R882H mutation in leukaemogenesis we generated a novel, inducible knock‐in mouse line of human DNMT3A‐R882H mutations (DNMT3AR882H). DNMT3AR882H knock‐in mice that harbored a floxed stop cassette upstream of the Dnmt3a gene were crossed with mice carrying an interferon‐inducible Cre recombinase allele (Mx1‐Cre). Upon Cre induction the endogenous mouse Dnmt3a gene from exon 2 was efficiently replaced by human DNMT3A cDNA carrying the R882H mutation.Results:Analysis of aged DNMT3AR882H knock‐in mice at pre‐leukemic stage revealed accumulation of myeloid progenitor (GMP) and stem cells (HSC), which showed loss of dormancy, increased cell cycle and exhaustion in serial retransplantation in vivo. Using mouse model of leukemia by mouse‐intercross and retroviral infection, we have confirmed that DNMT3AR882H mutation accelerated leukaemogenesis in the presence of FLT3‐ITD, MLL‐AF9 and Myc‐Bcl2, whereas DNMT3AR882H mutation alone did not induce leukemia in mice up to eighteen months after Cre induction.In search of therapeutic strategies, we identified that DNMTi specifically target DNMT3A‐R882H mutant pre‐leukemic HSC and leukemic blasts. Loss of DNA methylation in DNMT3A‐R882H HSCs associated with enhanced immune signals including increased expression of endogenous retrovirus (ERV) transcripts, increased cytosolic dsRNA, and activation of an IFN‐inducing cellular response. A single therapy cycle of low dose DNMTi (5‐Azacytidine) treatment accelerated loss of DNA methylation in DNMT3A‐R882H HSCs and exhaustion of DNMT3A‐R882H mutant hematopoiesis. 5‐Azacytidine in combination with chemotherapy induced long lasting remissions in DNMT3A‐R882H murine leukemias.Summary/Conclusion:These findings may open the path towards targeted therapy of DNMT3A mutated clonal hematopoiesis and AML.