Conditional Knockout Allele Research Articles

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.

GATA2 functions in adrenal chromaffin cells.

Catecholamine synthesized in the sympathoadrenal system, including sympathetic neurons and adrenal chromaffin cells, is vital for cardiovascular homeostasis. It has been reported that GATA2, a zinc finger transcription factor, is expressed in murine sympathoadrenal progenitor cells. However, a physiological role for GATA2 in adrenal chromaffin cells has not been established. In this study, we demonstrate that GATA2 is specifically expressed in adrenal chromaffin cells. We examined the consequences of Gata2 loss-of-function mutations, exploiting a Gata2 conditional knockout allele crossed to neural crest-specific Wnt1-Cre transgenic mice (Gata2 NC-CKO). The vast majority of Gata2 NC-CKO embryos died by embryonic day 14.5 (e14.5) and exhibited a decrease in catecholamine-producing adrenal chromaffin cells, implying that a potential catecholamine defect might lead to the observed embryonic lethality. When intercrossed pregnant dams were fed with synthetic adrenaline analogs, the lethality of the Gata2 NC-CKO embryos was partially rescued, indicating that placental transfer of the adrenaline analogs complements the lethal catecholamine deficiency in the Gata2 NC-CKO embryos. These results demonstrate that GATA2 participates in the development of neuroendocrine adrenaline biosynthesis, which is essential for fetal survival.

Loss of CPAP in developing mouse brain and its functional implication for human primary microcephaly.

Primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by small brain size with mental retardation. CPAP (also known as CENPJ), a known microcephaly-associated gene, plays a key role in centriole biogenesis. Here, we generated a previously unreported conditional knockout allele in the mouse Cpap gene. Our results showed that conditional Cpap deletion in the central nervous system preferentially induces formation of monopolar spindles in radial glia progenitors (RGPs) at around embryonic day 14.5 and causes robust apoptosis that severely disrupts embryonic brains. Interestingly, microcephalic brains with reduced apoptosis are detected in conditional Cpap gene-deleted mice that lose only one allele of p53 (also known as Trp53), while simultaneous removal of p53 and Cpap rescues RGP death. Furthermore, Cpap deletion leads to cilia loss, RGP mislocalization, junctional integrity disruption, massive heterotopia and severe cerebellar hypoplasia. Together, these findings indicate that complete CPAP loss leads to severe and complex phenotypes in developing mouse brain, and provide new insights into the causes of MCPH.

Abstract A22: Kras drives changes in acinar-specific gene regulatory networks in early pancreatic neoplasia in conjunction with Bmi1

Abstract The initial stages of pancreatic ductal adenocarcinoma (PDA) involve an oncogenic Kras-driven metaplastic transition of acinar cells to a more duct-like phenotype referred to as acinar to ductal metaplasia (ADM). The exact mechanism by which Kras reprograms acinar cells to a more duct-like cell at the genomic level is not completely understood. Developmental transcription factor (TF) networks regulate and maintain the fate of cells in other contexts and rely on appropriate chromatin context for their activity. Deletion of Bmi1, a component of the Polycomb Repressor Complex 1, completely represses Kras-driven ADM and neoplasia in genetically engineered mouse models of PDA. Thus, we hypothesized that Kras reprograms the acinar fate TF network to promote PDA in a Bmi1-dependent epigenetic context. We used a genetically engineered mouse model of early pancreatic neoplasia bearing an acinar cell-specific tamoxifen inducible Cre-recombinase under the elastase promoter (Ela-CreER) combined with the Kras G12D oncogenic allele (Kras−LSL-G12D/+). We added the conditional Bmi1 knockout allele (Bmi1−fl) and the Rosa26 tdTomato reporter to generate Ela-CreER, Kras−LSL-G12D/+, Bmi1−fl/fl, R26 tdTomato mice and their litter mate controls with and without KrasG12D and Bmi1. We activated the Cre-recombinase in 6- to 8-week-old mice with 5 daily tamoxifen gavages (4mg/day) prior to induction of acute pancreatitis by intraperitoneal caerulein administration (eight doses per day for two days) one week after the first tamoxifen dose. We used FACS to sort tdTomato+ cells one week after pancreatitis induction and analyzed RNA expression levels of 26 TFs, previously reported to play a role in pancreatic development, homeostasis and neoplasia. Expression levels of three lineage markers (amylase, elastase and cytokeratin 19) were also measured by quantitative RT-PCR. Tamoxifen gavage induced high tdTomato expression in the acinar compartment of Ela-CreER+ mice. RT-qPCR of tdTomato+ cells indicated a loss of acinar specific TFs in mice expressing mutant Kras one week after induction of pancreatitis. This is consistent with cells undergoing ADM. Genetic ablation of Bmi1 in the presence of mutant Kras restored elastase and amylase expression at the mRNA level. At the morphologic level, loss of Bmi1 also led to recovery of acinar cells from ADM and correlated with an increase in expression of three key acinar-specific fate TFs, Mist1, Hnf1a and Nr5a2. The mRNA expression levels of other acinar TFs like Pdx1 and Ptf1a/p48 did not recover with loss of Bmi1 and mutant Kras expression. Together, our data suggest that oncogenic Kras-driven ADM is controlled by changes in master TF gene regulatory networks. Bmi1 deletion leads to partial reprogramming of these networks to allow acinar cells to resist Kras-driven oncogenesis. Citation Format: Joyce K. Thompson, Jack Blumberg, Osama Alkahili, Howard C. Crawford, Marina Pasca di Magliano, Filip Bednar. Kras drives changes in acinar-specific gene regulatory networks in early pancreatic neoplasia in conjunction with Bmi1 [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 A22.

Abstract B12: DOCK1 as a novel target for controlling RAS-driven cancer cell survival and invasion

Abstract Oncogenic RAS contributes to malignant phenotypes of cancer cells by stimulating macropinocytosis-dependent nutrient uptake and promoting invasive migration. Because these cellular activities require remodeling of the actin cytoskeleton, which is regulated by the Rho family GTPase Rac, we hypothesized that molecules involved in Rac activation downstream of oncogenic RAS may be valuable targets for cancer therapy. DOCK1 is a Rac-specific guanine nucleotide exchange factor (GEF) implicated in malignancy of breast cancers and glioma. We show that genetic inactivation of endogenous DOCK1 in various RAS-transformed cells markedly suppressed their matrix invasion, macropinocytosis, and survival under glutamine-deprived conditions. We developed a selective inhibitor of DOCK1 that binds to the catalytic domain of DOCK1 and blocks its GEF activity. This inhibitor 1-(2-(3′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-2-oxoethyl)-5-pyrrolidinylsulfonyl-2(1H)-pyridone (TBOPP) blocked DOCK1-mediated invasion and macropinocytosis without impairing the immune-regulatory functions of its closely related protein DOCK2. Furthermore, TBOPP treatment suppressed cancer metastasis and growth in vivo in mice. To further assess the role of DOCK1 in RAS-driven cancers, we established the KPC mouse pancreatic ductal adenocarcinoma model bearing DOCK1 conditional knockout allele. Our results demonstrate that targeting DOCK1 could be an effective approach to treat RAS-driven cancers. Citation Format: Takehito Uruno, Yoshinori Fukui. DOCK1 as a novel target for controlling RAS-driven cancer cell survival and invasion [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 B12.

Loss of Cardiac Ferritin H Facilitates Cardiomyopathy via Slc7a11-Mediated Ferroptosis.

Maintaining iron homeostasis is essential for proper cardiac function. Both iron deficiency and iron overload are associated with cardiomyopathy and heart failure via complex mechanisms. Although ferritin plays a central role in iron metabolism by storing excess cellular iron, the molecular function of ferritin in cardiomyocytes remains unknown. To characterize the functional role of Fth (ferritin H) in mediating cardiac iron homeostasis and heart disease. Mice expressing a conditional Fth knockout allele were crossed with 2 distinct Cre recombinase-expressing mouse lines, resulting in offspring that lack Fth expression specifically in myocytes (MCK-Cre) or cardiomyocytes (Myh6-Cre). Mice lacking Fth in cardiomyocytes had decreased cardiac iron levels and increased oxidative stress, resulting in mild cardiac injury upon aging. However, feeding these mice a high-iron diet caused severe cardiac injury and hypertrophic cardiomyopathy, with molecular features typical of ferroptosis, including reduced glutathione (GSH) levels and increased lipid peroxidation. Ferrostatin-1, a specific inhibitor of ferroptosis, rescued this phenotype, supporting the notion that ferroptosis plays a pathophysiological role in the heart. Finally, we found that Fth-deficient cardiomyocytes have reduced expression of the ferroptosis regulator Slc7a11, and overexpressing Slc7a11 selectively in cardiomyocytes increased GSH levels and prevented cardiac ferroptosis. Our findings provide compelling evidence that ferritin plays a major role in protecting against cardiac ferroptosis and subsequent heart failure, thereby providing a possible new therapeutic target for patients at risk of developing cardiomyopathy.

Abstract A88: RUNX1-deficiency in luminal mammary epithelial cells leads to development of a unique type of immune “hot” ER+ mammary tumor

Abstract Genetic mutations in cancer cells play key roles in shaping the tumor immune microenvironment. RUNX1 is a transcription factor (TF) essential for estrogen receptor (ER)+ luminal mammary epithelial cells (MECs) and is mutated in a significant portion of human ER+ luminal breast cancers. It is also an important regulator of immune cells, raising an intriguing question as to whether its loss-of-function mutations found in ER+ breast cancers affect both MECs and immune cells. To address this, we established an in vivo mouse model by inducing loss of both RUNX1 and p53 in luminal MECs, upon intraductal injection of a Cre-expressing adenovirus under the control of the Keratin 8 promoter (Ad-K8-Cre) to mammary glands of female mice carrying their conditional knockout alleles. Compared to similarly injected wild-type (WT) control mice or mice with induced loss of p53 alone, induced loss of both led to hyperproliferation of Runx1/Trp53-null MECs and eventually development of mammary tumors after a long latency (a tumor-free period of ~9-13 months after Ad-K8-Cre injection). Characterization of these tumors revealed that they are ER+ mammary tumors with extensive leukocyte infiltration. CD45+ leukocytes in them constituted ~32-84% of total live tumor cells, which is unusual for a mouse cancer model. Among CD45+ cells, ~45% were macrophages and ~15% were CD3+ T cells. Among tumor-infiltrating myeloid cells (CD45+CD11b+), many were myeloid-derived suppressor cells (MDSCs). In addition, significant differential expression of PD-1 on CD3+ T cells (38.5% vs. 7.3%) and PD-L1 on CD45- cells (2.7% vs. 0.3%) was observed in these Runx1/Trp53-null tumors, when compared to WT controls. This immune profile suggests an immunosuppressive microenvironment in these tumors. The tumor immune phenotype induced by RUNX1-loss in our model is supported by genomic data from human breast cancers analyzed by the TIMER online tool (Li et al., Cancer Res 77(21):e108-e110). The TIMER analysis showed that in human breast cancers, levels of most infiltrating immune cell subtypes (e.g., macrophages, T cells) in RUNX1-mutated cases are higher than those of RUNX1-WT cases; in contrast, a similar analysis for GATA3-mutated cases exhibited the opposite. As both RUNX1 and GATA3 are key TFs for luminal MECs and their mutations are both found in luminal breast cancers, the data from both human cases and our mouse model suggest that RUNX1-mutated breast cancers may represent a unique, immune “hot” subtype of ER+ luminal breast tumors. ER+ breast cancers are typically immune “cold” and do not respond to immune checkpoint blockade (ICB) well. Our data suggest that RUNX1-mutated breast cancers may represent a subset of ER+ breast cancers that may respond to ICB-based therapy. Collectively, our data suggest that loss of RUNX1 promotes development of ER+ breast cancer and modulates the immune microenvironment; a better understanding of how these two processes are interconnected may provide novel strategies to treat breast cancer with RUNX1-mutation. Citation Format: Dongxi Xiang, Zhe Li. RUNX1-deficiency in luminal mammary epithelial cells leads to development of a unique type of immune “hot” ER+ mammary tumor [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A88.

Consistent Production of Mice with Conditional Knockout Alleles by CRISPR/Cas9-Mediated Genome Editing Using Two Guides/Two Oligos Approach

Gene targeting is extensively used to generate designer mouse mutants and to study gene function in vivo. Knockout mice that harbor a null allele in their germline provide appropriate genetic models of inherited diseases and often exhibit embryonic or early postnatal lethality. To study gene function in adult mice and in selected cell types, a refined strategy for conditional gene inactivation has been developed that relies on the DNA recombinase Cre and its recognition (loxP) sites. This process has traditionally relied on the complex process involving genome editing in embryonic stem (ES) cells despite its limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse ES cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN), which has been questioned by another report combining data from multiple transgenic cores. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of using this method in creating conditional targeted alleles in three genes, phosphatase and actin regulator 1 (Phactr1), apolipoprotein A-I (ApoA1), and actin-related protein T2 (Actrt2). Even though overall success rate was low—about 2.5%—we show that it’s possible to reliably generate conditional knockout alleles using CRISPR/Cas9 on a consistent basis.

Abstract C56: Bmi1 is widely expressed in acini and regulates Kras-driven transcription factor networks in early pancreatic neoplasia

Abstract Rare acinar cells expressing Bmi1, a component of the Polycomb Repressor Complex 1 (PRC1), represent a reservoir of cells contributing to pancreatic repair during injury and stress. This is marked by acinar-ductal metaplasia (ADM), a transition of acinar cells to a more duct-like phenotype, which is seen in pancreatitis and early stages of Kras-driven pancreatic neoplasia. The mechanism behind Kras-driven ADM at the genomic level is not completely understood. Developmental transcription factor (TF) networks regulate and maintain the fate of cells and rely on appropriate chromatin context for their activity. Deletion of Bmi1 represses Kras-driven ADM and neoplasia in genetically engineered mouse models (GEMMs) of PDA. Thus, we hypothesized that Kras reprograms the acinar fate TF network to promote PDA in a Bmi1-dependent epigenetic context. We used GEMMs to study the expression of Bmi1 in the acinar compartment and its role in early pancreatic neoplasia. We combined an acinar cell-specific tamoxifen-inducible Cre-recombinase under the elastase promoter (Ela-CreER) with the Kras G12D oncogenic allele and the conditional Bmi1 knockout allele and the Rosa26 tdTomato reporter to generate Ela-CreER, KrasLSL-G12D/+, Bmi1, R26 tdTomato mice and their littermate controls. We also generated Ela-CreER, Bmi1GFP, R26tdTomato and Bmi1-CreER, R26 tdTomato mice to quantify the fraction of acinar cells expressing Bmi1. We activated the Cre-recombinase in 6- to 8-week-old Ela-CreER mice with 5 tamoxifen gavages (4mg/day) prior to induction of acute pancreatitis by intraperitoneal caerulein administration (8 hourly injections x 2 days) one week after the first tamoxifen dose. We FACS sorted tdTomato+ cells from untreated mice (0 hr) and one week (168 hr) after pancreatitis induction and analyzed RNA levels of 8 TFs, reported to play a role in pancreatic development, homeostasis, and neoplasia. Expression levels of two lineage markers (amylase and elastase) were also measured by RT-qPCR. Lineage tracing in Bmi1-CreER and Ela-CreER, Bmi1GFP mice revealed expression of Bmi1 in vast majority of acinar cells. RT-qPCR of tdTomato+ cells in the Ela-CreER models indicated a loss of acinar-specific TFs in mice expressing mutant Kras one week after induction of pancreatitis consistent with cells undergoing ADM. Genetic ablation of Bmi1 in the presence of mutant Kras restored elastase and amylase mRNA levels. At the morphologic level, loss of Bmi1 also led to recovery of acinar cells from ADM and correlated with an increase in RNA expression of three key acinar-specific fate TFs—Mist1, Hnf1a, and Nr5a2. The mRNA levels of other key acinar TFs—Pdx1 and Ptf1a/p48—did not recover with the loss of Bmi1 in the presence of KrasG12D. Together, our data suggest that Bmi1 is expressed in the majority of acinar cells and regulates oncogenic Kras-driven ADM by altering master TF gene regulatory networks. Its deletion leads to partial reprogramming of these networks to allow acinar cells to resist Kras-driven oncogenesis. Citation Format: Joyce K. Thompson, Emily Wu, Osama Alkhalili, Howard C. Crawford, Marina Pasca di Magliano, Filip Bednar. Bmi1 is widely expressed in acini and regulates Kras-driven transcription factor networks in early pancreatic neoplasia [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C56.

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.

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation

BackgroundCRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method).ResultsWe re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach.ConclusionWe propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

Using CRISPR/Cas9 engineering to generate a mouse with a conditional knockout allele for the promyelocytic leukemia zinc finger transcription factor.

The promyelocytic leukemia zinc finger (PLZF) transcription factor mediates a wide-range of biological processes. Accordingly, perturbation of PLZF function results in a myriad of physiologic defects, the most conspicuous of which is abnormal skeletal patterning. Although whole body knockout of Plzf in the mouse (Plzf KO ) has significantly expanded our understanding of Plzf function in vivo, a conditional knockout mouse model that enables tissue or cell-type specific ablation of Plzf has not been developed. Therefore, we used CRISPR/Cas 9 gene editing to generate a mouse model in which exon 2 of the murine Plzf gene is specifically flanked (or floxed) by LoxP sites (Plzf f/f ). Crossing our Plzf f/f mouse with a global cre-driver mouse to generate the Plzf d/d bigenic mouse, we demonstrate that exon 2 of the Plzf gene is ablated in the Plzf d/d bigenic. Similar to the previously reported Plzf KO mouse, the Plzf d/d mouse exhibits a severe defect in skeletal patterning of the hindlimb, indicating that the Plzf f/f mouse functions as designed. Therefore, studies in this short technical report demonstrate that the Plzf f/f mouse will be useful to investigators who wish to explore the role of the Plzf transcription factor in a specific tissue or cell-type.

Setd2-Mediated Epigenetic Regulation of Erythropoiesis and HSC Maintenance in Mouse Models

SET domain containing 2 (SETD2) is a methyltransferase for histone 3 lysine 36 tri-methylation (H3K36me3). Using a conditional knockout allele (Setd2Δ/Δ), we recently demonstrated that their SLAM-hematopoietic stem cells (SLAM-HSCs) have intrinsic defects in maintaining quiescence and multi-lineage differentiation potential. Setd2Δ/Δ mice show decreased lymphoid and myeloid progenitors except increased pre-colony-forming-unit-erythroid (Pre CFU-E) and erythroblasts in bone marrow (BM); they suffer from lethal macrocytic anemia. A decreasing H3K36me3, but increasing H3K36me1 and H3K36me2, were observed in Setd2Δ/Δ mice. As a consequence, RNA polymerase II elongation is enhanced at a subset of genes, including Myc which encodes a transcriptional factor essential for erythropoiesis and HSC activation (Zhou Y., Haematologica, 2018). In the current study, we aim to investigate the detailed mechanisms of Setd2 deficiency in the intrinsic and extrinsic mechanisms of erythropoiesis and in the maintenance of HSC from definitive HSCs to adult.Erythropoiesis from the BFU-E/CFU-E stage to mature erythrocytes is sequentially regulated by an erythropoietin (EPO)-dependent early stage and macrophages-dependent late stage. To understand the function of Setd2 in erythroid differentiation, we generated two Setd2 conditional knockout mice: Setd2flox/flox/Vav1-Cre+ (Setd2Δ/Δ) and Setd2flox/flox/EpoR-Cre+ (Setd2Δ/Δ/EpoR). Setd2Δ/Δmice harbor a pan-hematopoietic deletion, while Setd2Δ/Δ/EpoR mice harbor an erythroid stage specific deletion from CFU-Es, proerythroblasts and basophilic erythroblasts. In Setd2Δ/ΔBM, the absolute number of basophilic erythroblasts and polychromatic erythroblasts were significantly increased but mature red blood cells were decreased. Surprisingly, Setd2Δ/Δ/EpoR mice showed normal proportions of erythroid blasts and did not suffer from anemia, suggesting that Setd2 is not required for the proliferation and differentiation of EPO-dependent erythroblasts and thereafter mature erythrocytes. Central macrophages are known to play a crucial role in terminal erythropoiesis by supplying the iron and engulfing the protruded nuclei from erythroblasts in the terminal maturation process. We then analyzed macrophages in Setd2Δ/Δ mice. The total F4/80+ macrophages were not changed, however, the proportion of type II macrophages (CD206+CD80-F4/80+) were significantly increased in Setd2Δ/Δ BM. Myc is a well-known factor for polarizing type II immune phenotypes. Our data suggest that Setd2 deficiency causes a shift of central macrophage to type II, which possibly impedes the terminal maturation of erythrocytes and causes macrocytic anemia, even with significantly increased BFU-E, CFU-E, and erythroblasts.Next, we addressed whether Setd2 was required for HSC maintenance from definitive HSCs to adult HSCs in an autonomous manner. It has been shown that genes regulating the maintenance of HSC at different developmental stages are diverse. To clarify in which developmental stages Setd2 is critical for HSC maintenance, we examined the HSC populations in Setd2Δ/Δ embryos at E14.5, and in Setd2Δ/Δ mice BM at 3 weeks (wks), 8 wks and 34-38 wks of age. HSC populations from fetal liver (FL) at E14.5 and BM at 3 wks were not different from Setd2flox/flox control littermates. Significant reductions in the absolute number of SLAM-HSCs, CD201+SLAM-HSCs and multipotent progenitors were observed at 8 wks, eventually HSCs were completely exhausted for mice at 34-38 wks. We further evaluated the HSC function in a competitive bone marrow transplantation assay. Similar levels of multi-lineage reconstitution were observed in recipient mice with Setd2Δ/Δ donor cells from FL or BM transplanted at 3 wks. However, Setd2Δ/Δ HSCs from 8 wk and 34 wk BM failed to support a long-term reconstitution and there is a complete absence of donor derived Setd2Δ/Δ LSKs. Altogether, our data suggest that Setd2 is required for HSC maintenance at adult stage, but not at fetal or neonatal stages.In summary, our study revealed a previously unknown extrinsic function of Setd2 in terminal erythroid maturation; and also identified Setd2 as a critical intrinsic regulator for adult HSC maintenance. Our findings may provide unique insights into the defects in erythroid lineages, such as anemia or erythrocytosis, and also epigenetic mechanism of adult HSC maintenance. DisclosuresNo relevant conflicts of interest to declare.

Utx Insufficiency Cooperates with Braf V600E in the Induction of Myeloma in Mice

Introduction:Dysfunction of epigenetic pathways has been frequently implicated in hematological malignancies. In multiple myeloma (MM), EZH2, a methyltransferase that induces histone H3 lysine 27 trimethylation (H3K27me3), acts as an oncogene as evidenced by its overexpression, which was found to be positively correlated with disease progression (Kalushkova et al. PloS One. 2010). We have shown that inhibition of both EZH2 and its homolog EZH1 is effective in eradicating MM cells in vitro and in vivo (Rizq et al. Clin Cancer Res. 2017). In addition, inactivating somatic mutations in UTX/KDM6A, an X-linked histone demethylase that removes di- and tri-methyl groups from H3K27, are found in 3 - 10% of MM patients (van Haaften et al. Nat Genet. 2009 and Pawlyn et al. Clin Cancer Res. 2016), indicating a tumor suppressive role for UTX, which has yet to be delineated. Up till now, no mouse model has been generated to test Utx insufficiency in post germinal center (GC) B cells and plasma cells. On the other hand, an activating mutation V600E in the BRAF kinase gene is closely associated with aggressive disease features such as extramedullary disease and shorter overall survival in MM patients (Andrulis et al. Cancer Discov. 2013) and could accelerate induction of myeloma in mice.Methods:To investigate whether loss of Utx cooperates with Braf V600E in myelomagenesis in mice, we generated and analyzed mice with conditional knock-out allele of Utx and/or knock-in allele of Braf V600E combined with Cγ1-Cre allele, in which Cre is activated by immunization in post GC B cells.Results:Loss of Utx and Braf V600E synergistically induced post GC B-cell lymphoma and plasma cell neoplasms in mice and significantly shortened the survival of mice compared with control mice and either allele alone. Utx-/-Braf V600E females succumbed to death earlier than Utx-/YBraf V600Emales and Utx-/+Braf V600Efemales. Of note, plasma cell neoplasms developed at a high frequency in Utx-/YBraf V600Emales and Utx-/+Braf V600Efemales and, less frequently, in Utx-/-Braf V600E females. Mice with plasma cell neoplasms showed expansion of CD138+ plasma cells in bone marrow as well as spleen and/or lymph nodes, exhibiting extramedullary disease. Loss of Utx alleles and expression of Braf V600E were confirmed by genomic PCR of plasma cells. Importantly, the clonality of plasma cells was demonstrated by genomic PCR detecting rearrangements of immunoglobulin heavy and light chain genes. In addition, M protein was detected by serum protein electrophoresis (SPEP) at a high frequency. Notably, we were able to establish murine myeloma cell lines from moribund compound mice. These cells readily engrafted in the bone marrow of NOG mice after transplantation and caused myeloma-associated phenotypes including paraplegia in recipient mice. Interestingly, Utx-/-Braf V600E cells were sensitive to dual inhibition of EZH2 and EZH1 but not to specific inhibition of EZH2 in culture. They also showed decreased susceptibility to proteasome inhibitors when compared with human MM cell lines. To gain insight into the changes in the transcriptional landscape following Utx loss, we performed RNA sequencing (RNA seq) and then gene set enrichment analysis (GSEA). We found positive enrichment of gene sets related to Myc, implying that Myc is one of the main drivers of myelomagenesis in our mouse model. In addition, gene sets related to MM were significantly enriched following Utx loss. We are now working on ChIP sequencing (ChIP seq) of UTX-related histone modifications to evaluate the epigenetic impact of Utx loss on myelomagenesis.Conclusion:Utx insufficiency cooperates with Braf V600E in the induction of myeloma in mice. Our mouse model is a promising tool for understanding the role of epigenetic dysregulation in the pathogenesis of MM and evaluating novel anti-myeloma agents. DisclosuresOkuno:Celgene: Research Funding. Tamaru:Nichirei Bioscience INC.: Research Funding; Takeda Pharmaceutical Company Limited: Speakers Bureau.

ESAM on the Cells of Endothelial Lineage Plays an Important Role in the Development of Definitive Hematopoiesis

Endothelial cell-selective adhesion molecule (ESAM) is lifelong marker of hematopoietic stem cells (HSCs) (Yokota Blood 2009) and endothelial cells (ECs) (Hirarta JBC 2001). Although we previously elucidated the functional importance of ESAM on HSCs in stress-induced hematopoiesis in adults (Sudo J Immunol 2012; PLoS One 2016), how ESAM affect hematopoietic development during fetal life remained unknown. To address this issue, we analyzed the fetuses of conventional ESAM knockout mice. Approximately half of the ESAM-null fetuses exhibited severe anemia and died after mid-gestation. RNA-sequence analyses exhibited downregulation of adult-type globin genes and Alas2, which encodes the first, rate-limiting enzyme in the heme biosynthesis pathway in the ESAM-null fetal liver (FL). Culture and transplantation experiments demonstrated that these abnormalities were attributable to the malfunctioning of ESAM-null HSCs. In addition, cross-linking ESAM with an anti-ESAM antibody influenced the transcription of hemoglobin synthesis-related genes in HSCs, indicating the significance of ESAMs on HSCs for the development of definitive hematopoiesis. However, it was unclear whether ESAM expression on only HSCs was critical or if ESAM expression on ECs was also involved. To examine this, we generated ESAM-flox mice using the innovative CLICK (CRISPR with lssDNA inducing conditional knockout alleles) method that was recently developed by our group (Miyasaka BMC Genomics 2018), and analyzed the results.We first tried to establish endothelial lineage-specific ESAM-conditional knockout (ESAM-cKO) mice by crossing ESAM-flox mice with Cre-ERT2-expressing mice under the control of the VE-cadherin gene promoter (Okabe Cell 2014). After peritoneally administering tamoxifen to pregnant females on embryonic day (E) 12.5, 14.5, or 15.5, the FLs were examined on E17.5. Analyses of these fetuses showed that, as observed in conventional ESAM-null mice, severe anemia occurred in approximately half of the Cdh5-BAC-CreERT2 ESAMflox/flox fetuses. However, we noticed that ESAM expression was almost completely deleted not only on ECs but also on HSCs of this model even after a single tamoxifen injection on E15.5.Next, we made use of another ESAM-cKO model obtained by crossing ESAM-flox mice with Cre recombinase-expressing mice under the control of the Vav gene promoter (Georgiades Genesis 2002). Although this Vav-Cre model has been widely used as a hematopoietic cell-specific gene knockout system, the EC lineage is affected by mouse-to-mouse variation (Joseph Cell Stem Cell 2013). Indeed, we observed that ESAM expression on FL ECs was suppressed with varying efficiency in each mouse, whereas ESAM expression on lineage negative Sca1+c-kitHi (LSK) CD48- cells was absent. Notably, the absolute number of LSK CD48- cells in FLs was positively correlated with the remaining levels of ESAM expression on ECs. Furthermore, the anemic phenotype was less severe and fetal mortality was less evident in Vav-Cre-induced ESAM-cKO fetuses. These results indicated that ESAM on ECs also play an important role in the normal development of definitive HSCs and erythrocytes in the FL. These findings were unexpected because we previously observed that the contribution of ESAM on non-hematopoietic cells to HSC maintenance was subtle in the adult bone marrow (BM) of transplantation-induced chimeric mice (Sudo PLoS One 2016). However, this contradiction might be explained by the difference between the FL and adult BM. Various types of cells have been reported to serve as the so-called “HSC-niche” in the adult BM, whereas most HSCs are located adjacent to sinusoidal ECs in the FL.In conclusion, our data strongly suggest that ESAM expression on ECs, as well as HSCs, plays an important role in the development of definitive hematopoiesis. ESAM is known to mediate cell-cell interaction through homophilic binding. The results showed that ESAM on ECs in the developing liver compensated for ESAM deficiency on HSCs to ameliorate the lethal anemic phenotype. This indicates that ESAM might interact with undetermined molecules on developing HSCs, and these molecules might have a functional overlap with ESAM. DisclosuresYokota:CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Pfizer Inc.: Research Funding; Bristol-Myers Squibb: Research Funding; MSD K.K.: Research Funding; Celgene: Research Funding. Ichii:Celgene K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Novartis Pharma K.K.: Speakers Bureau. Ezoe:TAIHO Phamaceutical Co., Ltd.: Research Funding. Shibayama:Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding; Jansen Pharmaceutical K.K: Honoraria; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Oritani:Novartis Pharma: Speakers Bureau. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

Multiple neurons encode CrebB dependent appetitive long-term memory in the mushroom body circuit.

Lasting changes in gene expression are critical for the formation of long-term memories (LTMs), depending on the conserved CrebB transcriptional activator. While requirement of distinct neurons in defined circuits for different learning and memory phases have been studied in detail, only little is known regarding the gene regulatory changes that occur within these neurons. We here use the fruit fly as powerful model system to study the neural circuits of CrebB-dependent appetitive olfactory LTM. We edited the CrebB locus to create a GFP-tagged CrebB conditional knockout allele, allowing us to generate mutant, post-mitotic neurons with high spatial and temporal precision. Investigating CrebB-dependence within the mushroom body (MB) circuit we show that MB α/β and α'/β' neurons as well as MBON α3, but not in dopaminergic neurons require CrebB for LTM. Thus, transcriptional memory traces occur in different neurons within the same neural circuit.

Depletion of SMC5/6 sensitizes male germ cells to DNA damage.

The structural maintenance of chromosomes complex SMC5/6 is thought to be essential for DNA repair and chromosome segregation during mitosis and meiosis. To determine the requirements of the SMC5/6 complex during mouse spermatogenesis we combined a conditional knockout allele for Smc5, with four germ cell–specific Cre-recombinase transgenes, Ddx4-Cre, Stra8-Cre, Spo11-Cre, and Hspa2-Cre, to mutate Smc5 in spermatogonia, in spermatocytes before meiotic entry, during early meiotic stages, and during midmeiotic stages, respectively. Conditional mutation of Smc5 resulted in destabilization of the SMC5/6 complex. Despite this, we observed only mild defects in spermatogenesis. Mutation of Smc5 mediated by Ddx4-Cre and Stra8-Cre resulted in partial loss of preleptotene spermatocytes; however, spermatogenesis progresses and mice are fertile. Mutation of Smc5 via Spo11-Cre or Hspa2-Cre did not result in detectable defects of spermatogenesis. Upon exposure to gamma irradiation or etoposide treatment, each conditional Smc5 mutant demonstrated an increase in the number of enlarged round spermatids with multiple acrosomes and supernumerary chromosome content. We propose that the SMC5/6 complex is not acutely required for premeiotic DNA replication and meiotic progression during mouse spermatogenesis; however, when germ cells are challenged by exogenous DNA damage, the SMC5/6 complex ensures genome integrity, and thus, fertility.

Abstract A07: Speedy mouse models to study melanomagenesis

Abstract Melanoma accounts for only about 1% of the skin cancer, but contributes to a vast majority of skin cancer deaths. Despite extensive progress in treating melanoma with immunotherapy and small-molecule inhibitors, melanoma patients still suffer from tumor recurrence, mainly due to inherent or acquired resistance to these treatments. A better understanding of the detailed molecular mechanisms that contribute to melanoma formation and resistance may provide novel avenues for the development of improved treatment strategies. Genetically engineered mouse models (GEMMs) closely recapitulate the genetics and etiology of human malignancies and have significantly contributed to our understanding of the mechanisms underlying melanoma initiation and progression. However, the generation and characterization of GEMMs is often challenging due to their time-consuming, laborious, and expensive nature. To overcome some of these limitations, we sought to develop a melanoma mouse-modeling platform, which allows for accelerated speed, increased throughput, and lower cost of in vivo melanoma studies compared to conventional mouse models. To this end, we established several embryonic stem cell (ESC) lines harboring multiple alleles to aide in creating versatile melanoma mouse models. These alleles include Cre-inducible endogenous BrafV600E or NrasQ61R alleles for melanoma initiation, conditional PTEN knockout alleles for melanoma progression, the melanocyte-specific, tamoxifen-inducible Tyr-CreERt2 allele, a Cags-LSL-rtTA3 allele to activate Tet-inducible expression cassettes, and the collagen homing cassette (CHC) for the efficient delivery of such expression cassettes via recombination-mediated cassette exchange (RMCE). We demonstrated that our newly derived ESCs can be targeted with various constructs via RMCE with extremely high efficiency, thus significantly reducing the number of clones one has to screen for successful targeting. Moreover, we confirmed pluripotency of our ESC lines by Oct4 and Nanog staining, and by demonstrating their ability to generate chimeras via blastocyst injection and completely ESC-derived mice via tetraploid complementation. Topical application of 4-hydroxytamoxifen (4-OHT) on chimeras carrying the BrafV600E and PTENFlox alleles resulted in the development of nevi and melanomas within a few weeks. Importantly, our melanoma ESC-GEMM approach is compatible with novel genetic tools including inducible shRNA and CRISPR, thus providing a versatile and modular platform for the study of gene function in melanomagenesis. Thus, we have established a speedy mouse-modeling platform that will stimulate and accelerate in vivo melanoma research, and will provide a useful resource to investigate the pathobiology of melanomagenesis and to develop and preclinically test novel treatments. Citation Format: Ilah Bok, Florian A. Karreth. Speedy mouse models to study melanomagenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A07.

CLICK: one-step generation of conditional knockout mice

BackgroundCRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive.ResultsHere, we show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats.ConclusionsThe high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice.

In vivo CRISPR screening unveils histone demethylase UTX as an important epigenetic regulator in lung tumorigenesis

Lung cancer is the leading cause of cancer-related death worldwide. Inactivation of tumor suppressor genes (TSGs) promotes lung cancer malignant progression. Here, we take advantage of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated somatic gene knockout in a KrasG12D/+ mouse model to identify bona fide TSGs. From individual knockout of 55 potential TSGs, we identify five genes, including Utx, Ptip, Acp5, Acacb, and Clu, whose knockout significantly promotes lung tumorigenesis. These candidate genes are frequently down-regulated in human lung cancer specimens and significantly associated with survival in patients with lung cancer. Through crossing the conditional Utx knockout allele to the KrasG12D/+ mouse model, we further find that Utx deletion dramatically promotes lung cancer progression. The tumor-promotive effect of Utx knockout in vivo is mainly mediated through an increase of the EZH2 level, which up-regulates the H3K27me3 level. Moreover, the Utx-knockout lung tumors are preferentially sensitive to EZH2 inhibitor treatment. Collectively, our study provides a systematic screening of TSGs in vivo and identifies UTX as an important epigenetic regulator in lung tumorigenesis.