Knock-in Allele Research Articles

Abstract 1543: Development of a novel ECAD-EmGFP reporter line for pancreatic cancer MET study and drug discovery

Abstract World-wide, metastasis continues to be the leading cause of death in cancer patients1. Although epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) have been implicated in the progression of cancer metastasis and drug resistance, their mechanistic pathway and specific role in disease progression is not fully understood2. As such, the development of a reporter line that enables the real-time monitoring of the changing status of cells will not only aid in dissecting the EMT/MET pathway in the research field, but could also become a robust platform for new cancer drug development. E-cadherin, an adhesion protein expressed in epithelial cells, is upregulated in pancreatic cancer cells during MET and is associated with an increase in tight junctions and apico-basal polarity, as well as a change in morphology3,4. Here, we developed a novel PANC-1 ECAD-EmGFP reporter line using CRISPR/Cas9 genome-editing technology. In this cell line, the emerald green fluorescent protein (EmGFP) reporter was incorporated into the last exon of the endogenous E-cadherin gene, enabling real-time monitoring of MET status in live cells. The ECAD-EmGFP knock-in allele was confirmed at genomic, transcriptional, and translational levels. Functional data revealed that miRNA-200 treatment induced the increased expression of ECAD-EmGFP, and decreased expression of Snail, a transcription factor associated with mesenchymal traits. These changes in marker gene expression, as well as the decrease in invasive capacity upon induction, suggest that cells have undergone MET. This cell line is a valuable tool for dissecting the molecular mechanisms underlying EMT and MET and for evaluating or screening compounds targeting EMT and MET in pancreatic cancer. 1. Dizon DS, et al. Clinical Cancer Advances 2016: Annual Report on Progress Against Cancer from the American Society of Clinical Oncology. J Clin Oncol 34: 987-1011, 2016. 2. Nieto MA, et al. EMT: 2016. Cell 166: 21-45, 2016. 3. Polireddy K, et al, Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation. PLOS ONE 11(10): e0164811, 2016 4. Wong S, et al. E-cadherin: Its dysregulation in carcinogenesis and clinical implications. Critical Reviews in Oncology / Hematology 121: 11- 22, 2018 Citation Format: Diana Douglas, Sangeeta Kumari, Jarkko Huuskonen, Weiguo Shu. Development of a novel ECAD-EmGFP reporter line for pancreatic cancer MET study and drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1543.

Development and patterning of rib primordia are dependent on associated musculature

The importance of skeletal muscle for rib development and patterning in the mouse embryo has not been resolved, largely because different experimental approaches have yielded disparate results. In this study, we utilize both gene knockouts and muscle cell ablation approaches to re-visit the extent to which rib growth and patterning are dependent on developing musculature. Consistent with previous studies, we show that rib formation is highly dependent on the MYOD family of myogenic regulatory factors (MRFs), and demonstrate that the extent of rib formation is gene-, allele-, and dosage-dependent. In the absence of Myf5 and MyoD, one allele of Mrf4 is sufficient for extensive rib growth, although patterning is abnormal. Under conditions of limiting MRF dosage, MyoD is identified as a positive regulator of rib patterning, presumably due to improved intercostal muscle development. In contrast to previous muscle ablation studies, we show that diphtheria toxin subunit A (DTA)-mediated ablation of muscle progenitors or differentiated muscle, using MyoDiCre or HSA-Cre drivers, respectively, profoundly disrupts rib development. Further, a comparison of three independently derived Rosa26-based DTA knockin alleles demonstrates that the degree of rib perturbations in MyoDiCre/+/DTA embryos is markedly dependent on the DTA allele used, and may in part explain discrepancies with previous findings. The results support the conclusion that the extent and quality of rib formation is largely dependent on the dosage of Myf5 and Mrf4, and that both early myotome-sclerotome interactions, as well as later muscle-rib interactions, are important for proper rib growth and patterning.

The SLC36 transporter Pathetic is required for neural stem cell proliferation and for brain growth under nutrition restriction

BackgroundDrosophila neuroblasts (NBs) are neural stem cells whose maintenance relies on Notch activity. NBs proliferate throughout larval stages to generate a large number of adult neurons. Their proliferation is protected under conditions of nutrition restriction but the mechanisms responsible are not fully understood. As amino acid transporters (Solute Carrier transporters, SLCs), such as SLC36, have important roles in coupling nutrition inputs to growth pathways, they may have a role in this process. For example, an SLC36 family transporter Pathetic (Path) that supports body size and neural dendrite growth in Drosophila, was identified as a putative Notch target in genome-wide studies. However, its role in sustaining stem cell proliferation and maintenance has not been investigated. This study aimed to investigate the function of Path in the larval NBs and to determine whether it is involved in protecting them from nutrient deprivation.MethodsThe expression and regulation of Path in the Drosophila larval brain was analysed using a GFP knock-in allele and reporter genes containing putative Notch regulated enhancers. Path function in NB proliferation and overall brain growth was investigated under different nutrition conditions by depleting it from specific cell types in the CNS, using mitotic recombination to generate mutant clones or by directed RNA-interference.ResultsPath is expressed in both NBs and glial cells in the Drosophila CNS. In NBs, path is directly targeted by Notch signalling via Su(H) binding at an intronic enhancer, PathNRE. This enhancer is responsive to Notch regulation both in cell lines and in vivo. Loss of path in neural stem cells delayed proliferation, consistent with it having a role in NB maintenance. Expression from pathNRE was compromised in conditions of amino acid deprivation although other Notch regulated enhancers are unaffected. However, NB-expressed Path was not required for brain sparing under amino acid deprivation. Instead, it appears that Path is important in glial cells to help protect brain growth under conditions of nutrient restriction.ConclusionsWe identify a novel Notch target gene path that is required in NBs for neural stem cell proliferation, while in glia it protects brain growth under nutrition restriction.

Bi-FoRe: an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.

Endothelin signaling promotes melanoma tumorigenesis driven by constitutively active GNAQ.

The G-protein-coupled receptor, endothelin receptor B (EDNRB), is an important regulator of melanocyte survival and proliferation. It acts by stimulating downstream heterotrimeric G proteins, such as Gαq and Gα1 . Constitutively active, oncogenic versions of Gαq and Gα11 drive melanomagenesis, but the role of Ednrb in the context of these mutant G proteins has not been previously examined. In this paper, we used a knock-in mouse allele at the Rosa26 locus to force oncogenic GNAQQ209L expression in melanocytes in combination with Ednrb gene knockout. The resulting pathological analysis revealed that every aspect of melanomagenesis driven by GNAQQ209L was inhibited. We conclude that even in the presence of oncogenic Gαq , the Ednrb receptor activates normal Gαq and Gα11 proteins. This likely promotes tumorigenesis by activating phospholipase C-beta, the immediate effector of Gαq/11 . These findings suggest that it might be possible to target upstream receptors to offset the effects of hyperactive G proteins, recognized as the cause of a growing number of human disorders.

Systemic but not MDSC-specific IRF4 deficiency promotes an immunosuppressed tumor microenvironment in a murine pancreatic cancer model

Pancreatic ductal adenocarcinoma is characterized by a strong immunosuppressive network with a dense infiltration of myeloid cells including myeloid-derived suppressor cells (MDSC). Two distinct populations of MDSC have been defined: polymorphonuclear MDSC (PMN-MDSC) and monocytic MDSC (M-MDSC). Several factors influence the development and function of MDSC including the transcription factor interferon regulatory factor 4 (IRF4). Here, we show that IRF4 deficiency accelerates tumor growth and reduces survival, accompanied with a dense tumor infiltration with PMN-MDSC and reduced numbers of CD8+ T cells. As IRF4 has been described to modulate myeloid cell development and function, particularly of PMN-MDSC, we analyzed its role using MDSC-specific IRF4 knockout mice with the Ly6G or LysM knock-in allele expressing Cre recombinase and Irf4flox. In GM-CSF-driven bone marrow cultures, IRF4 deficiency increased the frequency of MDSC-like cells with a strong T cell suppressive capacity. Myeloid (LysM)-specific depletion of IRF4 led to increased tumor weight and a moderate splenic M-MDSC expansion in tumor-bearing mice. PMN cell (Ly6G)-specific depletion of IRF4, however, did not influence tumor progression or MDSC accumulation in vivo in accordance with our finding that IRF4 is not expressed in PMN-MDSC. This study demonstrates a critical role of IRF4 in the generation of an immunosuppressive tumor microenvironment in pancreatic cancer, which is independent of IRF4 expression in PMN-MDSC.

SUN-646 A Novel Population of FOXO1-Expressing Cells in the Stomach Controls Cell Plasticity by Regulating the Cyclin CCNE1

A promising new therapy for type 1 diabetes is the reprogramming of gut enteroendocrine cells into cells that produce insulin. The mechanism by which gut epithelial cells are converted into cells that make insulin remains unknown. We have previously found that elimination of Foxo1 in neurogenin3 (Ngn3)-expressing cells of the intestine generates glucose-sensing, insulin-producing cells that are capable of reversing streptozotocin-induced diabetes. Others have reported that stomach cells have a similar property when made to express β-cell factors Ngn3, Pdx1, and MafA. Using mice bearing a Foxo1-GFP knock-in allele, we traced Foxo1-expressing cells in the gut to subpopulations of Ngn3+, as well as acid-secreting parietal stomach cells. To study these cells, we established a 2D co-culture method in which primary stomach cells are isolated from mice and cultured with embryonic fibroblasts. Deletion of Foxo1 in this system generated cells immunoreactive for insulin and C-peptide. Interestingly, Foxo1 ablation also altered the abundance of other gastric cell populations, including more parietal cells and decreased expression of stem cell marker, Lgr5. Tissue-specific elimination of Foxo1 in vivo in either Ngn3+ or parietal cells also resulted in the appearance of insulin+ cells, increased parietal cells, and reduced Lgr5 mRNA. To determine how Foxo1 regulated these changes, we used cells isolated from reporter mice that change from red to green after genetic recombination to collect Foxo1-deleted primary stomach cells using FACS. While the mRNA levels of many known Foxo1 targets did not change, cyclin E1 (CCNE1), which regulates G1 to S-phase progression of the cell cycle, was significantly decreased. Conversely, primary stomach cells overexpressing Foxo1 had increased levels of CCNE1. Finally, using ChIP-seq, we found that Foxo1 binds directly to the CCNE promoter in a nutrient-dependent manner. In summary, we show that Foxo1 is expressed in a subpopulation of stomach parietal cells and that it regulates their function through the cell cycle regulator, CCNE1.

Dysregulated TGF-β Signaling Leads to Cell-Intrinsic Changes in T cell Development that Promote Allergic Inflammation

Abstract Variants in genes in the Transforming Growth Factor Beta (TGFb) signaling pathway are associated with allergic diseases suggesting that this cytokine may be a central player in allergic disease. The role of TGFb in preserving T cell mediated self-tolerance is well-appreciated but how disruption in this pathway promotes allergic inflammation is not clear. TGFb signals through its receptor TGFβRI and II, leading to phosphorylation and nuclear translocation of Smad proteins 2 and 3. We previously demonstrated that patients with Loeys-Dietz Syndrome (LDS), an autosomal dominant disorder caused by mutations in TGFBR1 and TGFBR2 are highly predisposed to developing allergic disease. Both LDS patients and mice harboring a knock-in allele (Tgfbr1mut) of an LDS mutation known to cause severe disease in humans exhibit increased levels of total and food-specific IgE, consistent with their allergic tendencies. These serologic changes were associated with increased numbers of T follicular helper (Tfh) cells which promote memory B cell and plasma cell formation, and decreased T follicular regulatory (Tfr) cells, which suppress humoral immune responses, in LDS patients and mice. Using a mixed bone marrow chimera model we demonstrated that these alterations in T cell development are cell-intrinsic. Furthermore, OVA specific OTII cells harboring the Tgfbr1mut were more likely to accumulate in the peyer’s patch, differentiate into Tfh cells, and less likely to upregulate Foxp3 in response to orally ingested OVA. Tgfbr1mut OTII cells also produced more IL-4 and less IL-10 in response to OVA. These findings suggest that TGF-β signaling plays an important role in T cell development and function that, when disrupted, promotes allergic inflammation.

Abstract IA31: Genetic approaches for testing candidate drug targets for Ras-induced tumorigenesis in vivo

Abstract Despite remarkable progress over the past few years, developing chemical inhibitors that directly and selectively target mutant Ras proteins remains a fundamental challenge. Given this, inhibitors of proteins that function upstream or downstream of oncogenic Ras are a promising alternative therapeutic strategy for RAS mutant tumors. However, the complexity of Ras signaling networks and adaptive mechanisms that result in pathway “rewiring” complicates identifying genes and proteins that are essential for the growth and survival of these cancers. Genetically engineered mice that express mutant Ras alleles from the endogenous locus are a robust system for uncovering pathway dependencies and potential therapeutic vulnerabilities. We have generated novel strains of mice to address two specific questions. First, we collaborated with Kevin Haigis to generate a “second site” C181S substitution within a conditional NrasLSL-G12D mutant allele (Nat Genet 2008;40:600) to ask if palmitoylation is essential for myeloid transformation in vivo. We intercrossed mice carrying this mutation with the Mx1-Cre strain and induced Cre recombinase expression in the hematopoietic compartment by injecting compound mutant pups with polyI:polyC. As previously reported, homozygous NrasLSL-G12D mice uniformly developed an aggressive myeloproliferative disorder (MPD) characterized by markedly elevated blood leukocyte counts, splenomegaly, and death by age 9 months. By contrast, homozygous NrasLSL-G12D,C181S mice remained healthy with no evidence of hematologic disease despite constitutively elevated levels of RasGTP in bone marrow cells that were similar to those observed in NrasLSL-G12D mice. Whereas normal NRas and N-RasG12D proteins are predominantly in the plasma membrane, N-RasG12D, C181S was largely mis-localized in the cytosol. We also generated Mx1-Cre, NrasG12D,C181S/G12D compound heterozygous mutant mice to investigate potential interactions between oncogenic NrasG12D alleles with and without the C181S substitution. Over half of these mice developed aggressive hematologic malignancies, albeit with prolonged latency compared to homozygous NrasG12D mice. Importantly, genetic analysis of diseased tissues revealed reduced frequency or complete absence of the NrasG12D,C181S allele due to secondary somatic genetic events, which indicates strong selective pressure to overcome the growth-suppressive properties of unpalmitoylated N-Ras. Together, these studies validate the palmitoylation/depalmitoylation cycle as a therapeutic target in NRAS mutant hematologic malignancies and establish a novel model system for addressing this question in other cancers. In another ongoing project, we collaborated with Pedro Perez-Macera and David Tuveson to modify a LSL-KrasG12D knock-in allele to introduce a second-site amino acid Y64G substitution that severely impairs binding to PI3 kinase. Surprisingly, mice expressing KrasG12D,Y64G in the germline are viable and phenotypically normal, although they are born at a lower than expected Mendelian frequency. Whereas Mx1-Cre, KrasG12D/+ mice uniformly develop aggressive MPD and die by 4 months of age, KrasG12D,Y64G animals have normal blood counts at 12-18 months of age. However, 100% of KrasG12D,Y64G mice develop lung lesions by 1 year of age, and ultimately succumb from lung tumors with a median survival of 496 days. The majority of these lesions are classified pathologically as atypical lymphoid proliferation or papillary adenomas, with a few mice developing adenocarcinomas. These studies demonstrating that expressing a Y64G amino acid substitution in the context of oncogenic KrasG12D rescues embryonic lethality, abrogates myeloid disease, and attenuates lung tumorigenesis are generally consistent with and also extend elegant studies of Pi3kca mutant mice from the Downward lab (Cell 2007;129:957; Cancer Cell 2013;24:617). Beyond the bone marrow and lung, KrasG12D,Y64G mice are a potent genetic tool for dissecting the role of aberrant PI3 kinase signaling in lung, pancreatic, colon, and other tissues characterized by tumors driven by somatic KRAS mutations, and these data have implications for treating human cancers with KRAS mutations. Citation Format: Kevin M. Shannon. Genetic approaches for testing candidate drug targets for Ras-induced tumorigenesis in vivo [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA31.

Pronuclear Microinjection during S-Phase Increases the Efficiency of CRISPR-Cas9-Assisted Knockin of Large DNA Donors in Mouse Zygotes

In CRISPR-Cas9-assisted knockin (KI) in zygotes, a remaining challenge is routinely achieving high-efficiency KI of large (kilobase-sized) DNA elements. Here, we focus on the timing of pronuclear injection and establish a reliable homologous recombination (HR)-based method to generate large KIs in zygotes compared with two other types of KI strategies involving distinct DNA repair pathways. At the ROSA26 locus, pronuclear injection with CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9 protein at the S phase by using the HR-based method yields the most efficient and accurate KIs (up to 70%). This approach is also generally effective for generating large KI alleles at other gene loci. We further apply our method to efficiently obtain biallelic ROSA26 KIs by sequential injection into both pronuclei. Our results suggest that delivery of genome editing components and donor DNA into S-phase zygotes is critical for efficient KI of large DNA elements.

Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes.

Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.

Demarcation of Topologically Associating Domains Is Uncoupled from Enriched CTCF Binding in Developing Zebrafish.

SummaryCCCTC-binding factor (CTCF) is a conserved architectural protein that plays crucial roles in gene regulation and three-dimensional (3D) chromatin organization. To better understand mechanisms and evolution of vertebrate genome organization, we analyzed genome occupancy of CTCF in zebrafish utilizing an endogenously epitope-tagged CTCF knock-in allele. Zebrafish CTCF shares similar facets with its mammalian counterparts, including binding to enhancers, active promoters and repeat elements, and bipartite sequence motifs of its binding sites. However, we found that in vivo CTCF binding is not enriched at boundaries of topologically associating domains (TADs) in developing zebrafish, whereas TAD demarcation by chromatin marks did not differ from mammals. Our data suggest that general mechanisms underlying 3D chromatin organization, and in particular the involvement of CTCF in this process, differ between distant vertebrate species.

A non-canonical role for p27Kip1 in restricting proliferation of corneal endothelial cells during development.

The cell cycle regulator p27Kip1 is a critical factor controlling cell number in many lineages. While its anti-proliferative effects are well-established, the extent to which this is a result of its function as a cyclin-dependent kinase (CDK) inhibitor or through other known molecular interactions is not clear. To genetically dissect its role in the developing corneal endothelium, we examined mice harboring two loss-of-function alleles, a null allele (p27−) that abrogates all protein function and a knockin allele (p27CK−) that targets only its interaction with cyclins and CDKs. Whole-animal mutants, in which all cells are either homozygous knockout or knockin, exhibit identical proliferative increases (~0.6-fold) compared with wild-type tissues. On the other hand, use of mosaic analysis with double markers (MADM) to produce infrequently-occurring clones of wild-type and mutant cells within the same tissue environment uncovers a roughly three- and six-fold expansion of individual p27CK−/CK− and p27−/− cells, respectively. Mosaicism also reveals distinct migration phenotypes, with p27−/− cells being highly restricted to their site of production and p27CK−/CK− cells more widely scattered within the endothelium. Using a density-based clustering algorithm to quantify dispersal of MADM-generated clones, a four-fold difference in aggregation is seen between the two types of mutant cells. Overall, our analysis reveals that, in developing mouse corneal endothelium, p27 regulates cell number by acting cell autonomously, both through its interactions with cyclins and CDKs and through a cyclin-CDK-independent mechanism(s). Combined with its parallel influence on cell motility, it constitutes a potent multi-functional effector mechanism with major impact on tissue organization.

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.

Reveal the Neutrophil: Elucidating the Role of a Neutrophil-Specific JAK2-V617F Mutation

Introduction: Myeloproliferative neoplasms (MPN) are characterized by monoclonal proliferation of myeloid cells. The clinical presentation of classic BCR-ABL negative MPN includes blood count changes, splenomegaly, chronic inflammation and, prognostically relevant, thrombosis. Within polycythemia vera, essential thrombocythemia and primary myelofibrosis, the most frequent genetic alteration is the point mutation V617F (VF) in the Jak2 gene. Due to an activation of leucocyte β1 and β2 integrins, in particular on neutrophils, JAK2-VF is driving thrombus formation (Edelmann et al., JCI, 2018). Thus, understanding the role of neutrophils within JAK2-VF-positive MPN is of special interest. We developed a murine JAK2-VF Catchup model of MPN that is characterized by the neutrophil-specific expression of JAK2-VF in combination with the fluorescent protein tdTomato allowing in-vivo two-photon microscopy. Neutrophil-specificity was achieved through a knock-in allele of Cre recombinase and tdTomato replacing exon 1 of Ly6G (Hasenberg et al., Nat Methods, 2015). Methods: The murine JAK2-VF Catchup model was developed by crossing JAK2+/loxP-VF-loxP mice and Ly6G+/Cre-tdTomato CatchupIVM-red mice. Experiments were performed in 12 weeks old JAK2+/loxP-VF-loxP Ly6G+/Cre-tdTomato CatchupIVM-red (JAK2+/VF Catchup) mice (n=5) and JAK2+/+ Ly6G+/Cre-tdTomato CatchupIVM-red (JAK2+/+ Catchup) mice (n=5) as wildtype (WT) controls. Whole blood cell counts and spleen weight were determined. Bone marrow (BM) cells were harvested from hind limbs followed by isolation of granulocytes using negative selection. In order to determine hematopoietic stem and progenitor cells (HSPCs), BM and spleen cell suspensions were analyzed by flow cytometry (FC). Using ICAM-1, VCAM-1, E-selectin and P-selectin, each with and without Fc-tag, adhesion properties of the isolated granulocytes were examined by either FC or microplate reader analysis. The expression of β1 integrin was further analyzed by FC. Results and Discussion: JAK2+/VF Catchup mice showed a slight but significant increase in platelet counts [1544±58.02 Gpt/l; WT: 1190±90.50 Gpt/l; p=0.011], with the mean value still remaining within the normal range. Hematocrit, red blood cell, white blood cell and neutrophil count as well as spleen weight were also within the normal range showing no differences from their WT controls. Interestingly, neutrophil and platelet counts of JAK2+/VF Catchup mice significantly correlated with one another [p=0.0251; R2=0.8529]. MPN-typical changes in number and composition of c-Kit+ HSPCs in either BM or spleen could not be found in JAK2+/VF Catchup mice. Directly compared to reliably MPN-developing Vav-1-Cre JAK2+/VF mice, JAK2+/VF Catchup mice showed significantly less megakaryocyte-erythroid progenitor (MEP), Lin- Sca1+ c-Kit+ (LSK) and multipotent progenitor cells (MPP) in the BM. In spleen of JAK2+/VF Catchup mice, the fractions of Lin- c-Kit+ (LK) and granulocyte-macrophage progenitor cells (GMP) were significantly lower compared to Vav-1-Cre JAK2+/VF mice. Thus, the neutrophil-specific JAK2-VF mutation does not lead to apparent numerical or compositional changes of HSPCs. These results show that a neutrophil-specific JAK2-VF mutation is not sufficient to induce a characteristic phenotype of JAK2-VF-positive MPN. The expression of β1 integrin on granulocytes was significantly increased in JAK2+/VF Catchup mice [fold change vs. control; 1.126±0.041; WT: 1.0±0.011; p=0.0194]. Unexpectedly, despite increased β1 integrin expression, the binding of granulocytes from JAK2+/VF Catchup mice to either ICAM-1, VCAM-1, E-selectin or P-selectin corresponded to that of their WT controls. Static adhesion and soluble ligand binding assays showed similar results. Since adhesion of granulocytes appeared unchanged in-vitro, we hypothesize that expression of JAK2-VF in neutrophils is not sufficient to overactivate integrins but secondary extrinsic signals (e.g. cytokines) may play a dominant role. Further experimentation is under way to elucidate this molecular mechanism. Conclusions: In order to dissect the properties of JAK2-VF-positive neutrophils, the JAK2-VF Catchup model represents a promising murine model of MPN. Herewith, the initiation of thrombosis and the development of other granulocyte-driven disease manifestations of JAK2-VF induced MPN can be thoroughly examined. Disclosures No relevant conflicts of interest to declare.

Differential deletion of GDNF in the auditory system leads to altered sound responsiveness.

Glial-derived neurotrophic factor (GDNF) has been proposed as a potent neurotrophic factor with the potential to cure neurodegenerative diseases. In the cochlea, GDNF has been detected in auditory neurons and sensory receptor cells and its expression is upregulated upon trauma. Moreover, the application of GDNF in different animal models of deafness has shown its capacity to prevent hearing loss and promoted its future use in therapeutic trials in humans. In the present study we have examined the endogenous requirement of GDNF during auditory development in mice. Using a lacZ knockin allele we have confirmed the expression of GDNF in the cochlea including its sensory regions during development. Global inactivation of GDNF throughout the hearing system using a Foxg1-Cre line causes perinatal lethality but reveals no apparent defects during formation of the cochlea. Using TrkC-Cre and Atoh1-Cre lines, we were able to generate viable mutants lacking GDNF in auditory neurons or both auditory neurons and sensory hair cells. These mutants show normal frequency-dependent auditory thresholds. However, mechanoelectrical response properties of outer hair cells (OHCs) in TrkC-Cre GDNF mutants are altered at low thresholds. Furthermore, auditory brainstem wave analysis shows an abnormal increase of wave I. On the other hand, Atoh1-Cre GDNF mutants show normal OHC function but their auditory brainstem wave pattern is reduced at the levels of wave I, III and IV. These results show that GDNF expression during the development is required to maintain functional hearing at different levels of the auditory system.

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.

A reference map of murine cardiac transcription factor chromatin occupancy identifies dynamic and conserved enhancers

Mapping the chromatin occupancy of transcription factors (TFs) is a key step in deciphering developmental transcriptional programs. Here we use biotinylated knockin alleles of seven key cardiac TFs (GATA4, NKX2-5, MEF2A, MEF2C, SRF, TBX5, TEAD1) to sensitively and reproducibly map their genome-wide occupancy in the fetal and adult mouse heart. These maps show that TF occupancy is dynamic between developmental stages and that multiple TFs often collaboratively occupy the same chromatin region through indirect cooperativity. Multi-TF regions exhibit features of functional regulatory elements, including evolutionary conservation, chromatin accessibility, and activity in transcriptional enhancer assays. H3K27ac, a feature of many enhancers, incompletely overlaps multi-TF regions, and multi-TF regions lacking H3K27ac retain conservation and enhancer activity. TEAD1 is a core component of the cardiac transcriptional network, co-occupying cardiac regulatory regions and controlling cardiomyocyte-specific gene functions. Our study provides a resource for deciphering the cardiac transcriptional regulatory network and gaining insights into the molecular mechanisms governing heart development.

Controlled induction and targeted elimination of p16INK4a-expressing chondrocytes in cartilage explant culture.

Cellular senescence is a phenotypic state that contributes to age-related diseases through the secretion of matrix-degrading and inflammatory molecules. An emerging therapeutic strategy for osteoarthritis (OA) is to selectively eliminate senescent cells by initiating apoptosis. This study establishes a cartilage explant model of senescence induction and senolytic clearance using p16Ink4a expression as a biomarker of senescence. Growth-factor stimulation of explants increased the expression of p16Ink4a at both the mRNA and protein levels. Applying this culture system to cartilage from p16tdTom reporter mice (a knockin allele with tdTomato fluorescent protein regulated by the endogenous p16Ink4a promoter) demonstrated the emergence of a p16-high population that was quantified using flow cytometry for tdTomato. Cell sorting was used to separate chondrocytes based on tdTomato fluorescence and p16-high cells showed higher senescence-associated β-galactosidase activity and increased gene expression of the senescence-associated secretory phenotype as compared with p16-low cells. The potential for effective senolysis within the cartilage extracellular matrix was assessed using navitoclax (ABT-263). Navitoclax treatment reduced the percentage of p16-high cells from 17.9 to 6.1% (mean of 13 matched pairs; P < 0.001) and increased cleaved caspase-3 confirmed apoptotic activity. Together, these findings establish a physiologically relevant cartilage explant model for testing the induction and elimination of senescent chondrocytes, which will support investigations of senolytic therapy for OA.-Sessions, G. A., Copp, M. E., Liu, J.-Y., Sinkler, M. A., D'Costa, S., Diekman, B. O. Controlled induction and targeted elimination of p16INK4a-expressing chondrocytes in cartilage explant culture.

Optimized CRISPR/Cas9-mediated in vivo genome engineering applicable to monitoring dynamics of endogenous proteins in the mouse neural tissues

To analyze the expression, localization, and functional dynamics of target proteins in situ, especially in living cells, it is important to develop a convenient, versatile, and efficient method to precisely introduce exogenous genes into the genome, which is applicable for labeling and engineering of the endogenous proteins of interest. By combining the CRISPR/Cas9 genome editing technology with an electroporation technique, we succeeded in creating knock-in alleles, from which GFP (RFP)-tagged endogenous proteins are produced, in neurons and glial cells in vivo in the developing mouse retina and brain. Correct gene targeting was confirmed by single-cell genotyping and Western blot analysis. Several gene loci were successfully targeted with high efficiency. Moreover, we succeeded in engineering the mouse genome to express foreign genes from the endogenous gene loci using a self-cleaving 2A peptide. Our method could be used to monitor the physiological changes in localization of endogenous proteins and expression levels of both mRNA and protein at a single cell resolution. This work discloses a powerful and widely applicable approach for visualization and manipulation of endogenous proteins in neural tissues.