TRPV4 mRNA and protein expression was confirmed in the H9C2 and NRVM, whereas functional TRPV4 activity was assessed from Ca2+ influx response to a TRPV4 agonist GSK1016790A. TRPV4 functional expression was significantly enhanced during H/R. Furthermore, H/R increased the intracellular Ca2+ concentration ([Ca2+]i) and induced cell injury, which were reversed by HC-067047 but was further aggravated by GSK1016790A. Moreover, HC-067047 treatment significantly alleviated the increase of reactive oxygen species (ROS) generation, the depolarization of mitochondrial membrane potential (Δψm) and the opening of mitochondrial permeability transition pore (mPTP) during H/R. On the contrary, GSK1016790A exacerbated those effects. Meanwhile, increase in [Ca2+]i and ROS induced by activation of TRPV4 was almost abolished when cells were cultured in Ca2+-free medium. In addition, ROS scavenger NAC obviously reversed activation of TRPV4-induced changes of Δψm and mPTP opening. Finally, we confirmed the direct roles of TRPV4 on cardiac injury and ROS generation in murine model myocardial I/R in vivo. In conclusion, activation of TRPV4 induces Ca2+ influx in cardiomyocytes, with subsequent ROS release, depolarizing of Δψm, opening mPTP, inducing injury and TRPV4 has key roles during I/R via these pathways.Transient receptor potential vanilloid 4 (TRPV4) is highly expressed in heart and vessels and can be activated during myocardial ischemia/reperfusion (I/R). Recently, we found that treatment with a selective TRPV4 antagonist HC-067047 significantly reduced infarct size, decreased troponin T levels and improved cardiac function in murine model myocardial I/R. This study was undertaken to investigate the mechanism underlying TRPV4-mediated myocardial I/R injury. To mimic myocardial I/R injury, we established a hypoxia/reoxygenation (H/R) model in H9C2 cells and neonatal rat ventricle myocytes (NRVMs)  It is widely distributed in various organs and tissues including heart and vessels.6 Indeed, our previous study has demonstrated that treatment with a selective TRPV4 antagonist HC-067047 significantly reduced infarct size, decreased troponin T levels and improved cardiac function in murine model myocardial I/R.7 Increased activation of TRPV4 has been also observed in the hippocampus after cerebral I/R, whereas blocking TRPV4 attenuated I/R-induced brain injury.11 Furthermore, sustained activation of TRPV4 dose-dependently induces apoptosis of retinal ganglion cells and neuronal death in the hippocampus.13 Therefore, TRPV4 may be a promising target to prevent myocardial I/R injury, but the mechanism underlying TRPV4-mediated myocardial I/R injury is still unclear.During myocardial ischemia/reperfusion (I/R), TRPV4 may be overactivated by cytotoxic edema or the metabolites of arachidonic acid, and it thus involves in myocardial I/R injury.2+. Activation of TRPV4 elicits Ca2+ influx and increases the intracellular concentration of free Ca2+ ([Ca2+]i).14 Furthermore, recent studies have found that TRPV4 agonists enhances the production of reactive oxygen species (ROS) via Ca2+ influx in endothelial cells, urothelial cell, macrophages, as well as hippocampus.18 In addition, Ca2+ overload and high level of ROS can trigger depolarization of mitochondrial membrane potential (Δψm), opening of the mitochondrial permeability transition pore (mPTP) and result in cell death, which contribute to myocardial I/R injury.19 Therefore, we hypothesized that TRPV4 activation exacerbates myocardial I/R injury via Ca2+/ROS/mPTP pathway. To mimic myocardial I/R injury, we established a hypoxia/reoxygenation (H/R) model in H9C2 cells and neonatal rat ventricle myocytes (NRVMs) in vitro. Some of the results were confirmed in the murine model myocardial I/R in vivo.TRPV4 is highly permeable to Ca2+ influx in response to the specific TRPV4 agonist, GSK1016790A. GSK1016790A induced robust concentration-dependent  Ca2+ influx in H9C2, which was almost blocked by pretreatment with a selective TRPV4 antagonist, HC-067047 (1 μM) for 30 min , which can be reduced and enhanced by HC-067047  and GSK1016790A  in H9C2 , which was significantly blunted in the presence of HC-067047  and aggravated by GSK1016790 . Importantly, the increase [Ca2+]i induced by GSK1016790 during H/R was completely abolished or strongly reduced when cells were cultured in Ca2+-free medium, consistent with the concept that the [Ca2+]i induced by activation of TRPV4 was mainly from extracellular Ca2+ influx. Interestingly, HC-067047 and GSK1016790A did not influence [Ca2+]i under normoxic conditions  level and heart function in myocardial I/R injury in vivo  and 0.25% trypsin 1:250  for 8 consecutive 7–10 min treatment periods at 37 °C. Cells were washed and then resuspended in 4.5 g/l glucose DMEM supplemented with FBS, and 1% penicillin/streptomycin (v/v) and 100 μM bromodeoxyuridine  was added. AMVM were isolated from the heart of 8–12 weeks adult C57BL/6 mice using a langendorff apparatus. After the animals were anesthetized with ether, their hearts were removed and retrograde perfusion through the aorta by 1 mg/ml collagenase type II for 10–15 min. H9C2 and NRVM were treated with a TRPV4 selective agonist GSK1016790A  and a TRPV4 selective antagonist HC-067047  at the onset of reoxygenation.Both rat heart tissue-derived H9C2 cardiac myoblast cell line and CHO cell line from ATCC  were cultured in Dulbecco's modified Eagle's medium , supplemented with 15% fetal bovine serum  at 37 °C in a humidified atmosphere of 95% air and 5% CO2 and 5% CO2 gas mixture in a controlled hypoxic plastic chamber  and replacing the media with fresh l.5 g/l glucose DMEM  without serum for 6 h. Subsequently, the medium was replaced by the normal media and incubated in 95% air and 5% CO2 for 6- h reoxygenation. Normal groups were cultured in normoxic conditions for corresponding times.When NRVM and H9C2 cells reached 80% confluence, hypoxia was induced by replacing the air content with a 95% N38 Oligonucleotide sequences of primers specific for TRPV4 were TRPV4: 5′-CCCGAGAGAACACCAAGTTTG-3′ (forward), and 5′-GACCGTCATTGTTAAGCACAGTCT-3′ (reverse), and β-ACTIN: 5′-CGTTGACATCCGTAAAGACC-3′ (forward), 5′-TAGAGCCACCAATCCACACA-3′ (reverse). Amplified products were separated on 2% agarose gels in TAE buffer, visualized with 1 μg/ml ethidium bromide. The relative expression quantity 2-ΔΔCt value was calculated to compare the differences among groups. The result for each gene was obtained from at least six independent experiments.Total RNAs were extracted from cultured H9C2, NRVM and AMVM cells. And RT-PCR and qPCR was performed as described previously.38 The protein concentrations in the supernatants were measured using a BCA kit . Protein extracts (20 μg) were run on 10% sodium dodecyl sulfate-polyacrylamide electrophoresis gels, and 10 μl biotinylated protein ladder  loaded into a separate lane. Then, it was transferred to a nitrocellulose membrane by Electrophoresis System . After being blocked with 5% nonfat milk for 2 h, the membranes were incubated with the appropriate primary antibodies  at 4 °C overnight, followed by incubation with a goat anti-rabbit IgG-HRP secondary antibody  anti-biotin HRP-linked antibody  at a dilution of 1 : 2000. The protein expression levels were visualized using enhanced chemiluminescence method by Bio-Rad ChemiDoc XRS  and quantified by Image Lab Software. β-Actin as an internal reference.Total protein was extracted from the cultured H9C2, NRVM and AMVM as previous described.40 In brief, NRVM and H9C2 cells were loaded with 2 μM Fluo-4/AM  for 30 min, washed three times with HEPES buffer that contained (in mM) 130 NaCl, 4.7 KCl, 2.0 CaCl2,1.2 MgSO4, 1.2 KH2PO4, 10 HEPES and 11 glucose at pH 7.4. Cells in 96-wells plate were illuminated at 488 nm and fluorescence emissions at 525 nm were captured with the Enspire Multimode Plate Reader . GSK1016790A  was used to induce Ca2+ influx. In some experiments, cells were pretreated with 1 μM HC-067047 for 30 min. Changes in [Ca2+]i upon response to GSK1016790A were presented as relative changes (F/F0) or fold changes (ΔF/F0), where F is the fluorescence at intermediate Ca2+ levels, F0 is the average fluorescence before drug stimulation, ΔF is the mean fluorescence after GSK1016790A stimulation at steady-state minus F0. [Ca2+]i was calculated with the formula: [Ca2+]i =Kd × (F0−Fmin)/(Fmax−F0); where Kd is the dissociation constant (345 nM for fluo-4), Fmin is the fluorescence intensity of the indicator in the absence of Ca2+ and is obtained by adding a solution of 5 mM EGTA for 15 min, and Fmax is the fluorescence of the Ca2+ saturated indicator and is obtained by adding a solution of 0.1% Triton-X100 in 2.2 mM CaCl2 for 15 min.Intracellular calcium was measured as described previously.38 Annexin V and PI fluorescein staining kit  were utilized to measure apoptosis by following the manufacturer’s instruction. Apoptosis rate was evaluated by Flow Cytometry .Cell death was quantified by analyzing LDH activity with commercial kits . Cell viability was measured using the Cell Counting Kit-8  as previously described.μM) at 37 °C for 20 min in the dark. The fluorescence was detected by a fluorescent microscope or a Enspire Multimode Plate Reader  (488 nm excitation and 525 nm emission).ROS production in H9C2 was detected using the Reactive Oxygen Species Assay Kit . Briefly, following treatment as mentioned in the section ‘H/R model’ above, the cells were incubated with DCFH-DA (10 μm) of the left ventricle (LV) were incubated with 10 μM DHE for 30 min at 37 °C followed by washing with PBS for three times. Nuclei were counterstained with DAPI for 5 min at room temperature. Images were obtained using an Olympus BX-51 epifluorescence microscope . The numbers of DHE-positive nuclei was counted in five random fields in three nonconsecutive sections per heart at × 400 magnification, and expressed as a percentage of the total number of DAPI-stained nuclei.Changes in ROS from myocardium after I/R were detected with a fluorescent indicator dihydroethidium . In brief, transverse croysections  and aggregates  were detected with a fluorescent microscope or a Enspire Multimode Plate Reader. The ratio of aggregated JC-1 and monomeric JC-1 represented ΔΨm.Changes of mitochondrial membrane potential (Δψm) were measured by staining with JC-1 . After treatment, H9C2 cells were incubated with 5 μM calcein/AM and 2 mM CoCl2 at 37 °C for 30 min. After washing, cells were illuminated at 488 nm and an emission wavelength of 525 nm was captured with the Enspire Multimode Plate Reader every 30 s. Results were expressed as a percentage of normoxic group.mPTP opening was assayed by measuring calcein  fluorescence quenched by cobalt chloride , as previously reported. In brief, H9C2 cells were loaded with 2 41 Briefly, male C57BL/6 mice (22–23 g) were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg), and the heart was exposed through a left thoracotomy at the forth intercostal space. The slipknot was tied around the left anterior descending coronary artery (LAD) 2–3 mm from its origin under a surgical microscope and released after 30 min of ischemia to allow reperfusion. Sham-operated (sham) animals were subjected to the same surgical procedures except that the slipknot was not tied.Adult male C57BL/6 mice were obtained from VITAL RIVER , bred and maintained on a chow diet in a 12-h light/12-h dark environment at 25 °C in the Tongji Medical School Animal Care. All animal procedures were approved by the Institutional Animal Care and Use Committee, which is certified by Huazhong University of Science and Technology Committee on Animal Care. The surgical procedures of I/R were performed as previously described.β-cyclodextrin, Amresco) and saline. Vehicle mice were injected with the same volume of saline.The TRPV4 antagonist HC-067047 (10 mg/kg) was intraperitoneally injected beginning at 1 h after reperfusion and then injected every 8 h, and the TRPV4 agonist GSK1016790A (0.025 mg/kg) were administrated via jugular vein on the onset of reperfusion. The concentrations of the above-listed chemicals were selected based on previous reports. HC-067047 were first dissolved in DMSO, and then diluted in 0.9% NaCl solution. GSK1016790A was prepared in 1% DMSO/20% Captisol  in phosphate buffer at pH 7.4 at 37 °C for 10 min, then they were fixed with 10% formaldehyde for 24 h and photos were taken. Regions negative for Evans Blue staining  and negative for TTC  were calculated by a blinded observer using the computer-assisted planimetry function in ImageJ 6.0 . The myocardial infarct size was expressed as a percentage of IA over total AAR. Serum troponin T levels were evaluated as a biomarker for cardiac damage using a quantitative assay  as previously described.A Vevo 2100 high-resolution microimaging system with a 30 MHz transducer was used . Mice were anesthetized with 1.5% isoflurane and two-dimensional echocardiographic views of the mid-ventricular short axis and parasternal long axes were obtained. M-mode images were used to measure LV and LV EF and FS, which were acquired by a technician who was blinded to the treatment groups. Data analysis was performed using the Visualsonics data analysis suite.t-tests or one-way ANOVA followed by Bonferroni’s post-hoc test were performed to analyze differences with group comparison. Values of P<0.05 were considered statistically significant.All values are expressed as mean±S.E.M., and were analyzed for at least six independent experiments. Two-tailed


ATP7B mutant rabbits are a potential model for human WD disease with applications in pathological analysis, clinical treatment and gene therapy research.CRISPR/Cas9 has recently been developed as an efficient genome engineering tool. The rabbit is a suitable animal model for studies of metabolic diseases. In this study, we generated ATP7B site-directed point mutation rabbits to simulate a major mutation type in Asians (p. Arg778Leu) with Wilson disease (WD) by using the CRISPR/Cas9 system combined with single-strand DNA oligonucleotides (ssODNs). The efficiency of the precision point mutation was 52.94% when zygotes were injected 14 hours after HCG treatment and was significantly higher than that of zygotes injected 19 hours after HCG treatment (14.29%). The rabbits carrying the allele with mutant ATP7B died at approximately three months of age. Additionally, the copper content in the livers of rabbits at the onset of WD increased nine-fold, a level similar to the five-fold increase observed in humans with WD. Thus, the efficiency of precision point mutations increases when RNAs are injected into zygotes at earlier stages, and the  However, despite long-term attempts in many other species, mammalian germ line-transferred ESCs have been developed in only rats5. Another technique, somatic cell nuclear transfer (SCNT), was first used to clone sheep in 19976, and this method of generating adult animals from a single cell has succeeded in creating genetically manipulated animals in many species, including sheep7, pigs9, ferrets9 and rabbits10. However, these methods depend on complicated techniques that can be performed by only well-equipped labs, and a substantial amount of time is required, particularly to produce conditional gene knock-out (CKO) or precision point mutation animals. In recent years, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR/Cas9 gene editing systems have been developed as efficient genome engineering tools to generate gene knock-out (KO) animals via targeted nucleotide sequence cleavage and non-homologous end-joining (NHEJ) by microinjection into fertilized eggs17. Although new genetic manipulation techniques have potential disadvantages, they can provide a more convenient approach for generating CKO animals19 or repairing the mutated genome in adult animals via homologous recombination (HR) by large DNA fragments21.Genetically modified (GM) animals are powerful tools for studying gene functions and understanding the mechanisms of certain inherited human diseases. Various genetic alterations have been induced in mice since the 1980s through techniques such as fine mES cell culture22, Huntington disease23, cystic fibrosis24 or haemophilia25. In some hereditary diseases, gene functions are lost because of changes in amino acids. However, certain hereditary diseases are induced by enhanced or varied gene functions derived from SNPs27. These hereditary diseases cannot be simulated by KO animals. Therefore, precision point mutation animals produced by a special base knock-in (KI) are necessary. Rabbits are phylogenetically closer to primates than rodents, and they are physically large enough to permit non-lethal monitoring of physiological changes. Several transgenic or low-density lipoprotein receptor gene mutation rabbit models have been used for the study of lipoproteins and atherosclerosis because the lipid metabolism of rabbits is similar to that of humans29. Furthermore, several KO rabbits have been produced for different human hereditary diseases since new gene editing technologies were introduced32, and a reporter gene has been knocked into the ROSA26 locus in rabbits33. Given the extensive use of rabbits in research, the creation of precision point mutation rabbit lines is highly desirable.Sequence variations named SNPs are important for increasing genetic variability but may also lead to hereditary diseases, such as Wilson disease (WD)ATP7B gene36. More than 600 mutations causing WD have been described. Different countries feature different hotspots. A missense mutation (p. Arg778Leu) in exon 8 of ATP7B is a major mutation affecting approximately 20% of people in certain parts of Asia39.In humans, ATP7B is an important protein that is mainly expressed in hepatocytes and contributes to transmembrane transport of copper. The dysfunction of ATP7B contributes to WD, an autosomal recessive genetic disorder of copper metabolism caused by a mutation in the ATP7B gene were derived by microinjecting synthesized RNAs with ssODN donor sequences into zygotes, and the efficiency of homology-directed knock-in of point mutations was enhanced when RNAs were injected into younger zygotes.In this study, we used the CRISPR/Cas9 system to create a single amino acid substitute rabbit model for WD. The defined point mutations in the rabbit All experimental protocols, including animal care, the microinjection protocol and embryo transfer, met the approval guidelines (IACUC No. A-2015-002) established by the Ethics Committee of the School of Medicine of Shanghai Jiao Tong University.The pT7-cas9 vector and the gRNA cloning vector pCD-CAS were purchased from Biotechnologies Co., Ltd. . The template sgF0 amplified from pCD-CAS by primers P-sgF0F and T7R1  replacement, which results in a synonymous mutation in the ssODN. The ssODNs with the desired mutation and two 46 bp homologous arms were synthesized by Thermo Fisher Scientific, Inc. (PAGE purified)  were devised to target the sequence of exon 8 of rabbit ATP7B. The DNA sequences of sgRNA1F and sgRNA2R omitting NGG PAM were capped with a T7 promoter sequence, and they were named P-sg RNA1 and P- sg RNA2, respectively Table . A misseXba I and purified by isopropanol precipitation. The linearized plasmid was transcribed into mRNA in vitro using a mMESSAGE mMACHINE T7 Ultra Transcription kit . Cas mRNA was purified by lithium chloride precipitation according to the manufacturer’s instructions. DNA templates of sgRNA1F and sgRNA1R were generated from sgF0 via PCR amplification by using the forward primers P-sgRNA1F and P-sgRNA1R with the reverse primer T7R1. The two abovementioned templates were reamplified with the T7 primer and T7R1. The products of the second PCR were purified and used for in vitro RNA synthesis with a MEGAshort T7 high yield Transcription Kit . The sgRNAs were purified with a MEGAclear-96 Purification of Transcription Reactions Kit in a 96-well format . Cas9 mRNA and sgRNAs were dissolved in TE buffer .The pT7-cas9 plasmid was digested with in vitro was detected with a Guide-it sgRNA in vitro transcription and screening system kit . The sgRNA-targeted fragments were amplified by PCR from wild rabbit DNA with the primers r78JDF and r78JDR. The cleavage reaction containing 20 ng of the experimental sgRNA sample and 100 ng of the sgRNA-targeted fragments was incubated at 37 °C for 1 hr and terminated by incubation at 70 °C for 10 min. The entire 10 μL reaction was analysed on a 2% TBE agarose gel.The efficacy of cleavage of sgRNAs 40 and supplemented with 5% FBS before microinjection.Mature female rabbits were superovulated by injection of 100 IU pregnant mare stimulation gonadotropin  followed by 100 IU human chorionic gonadotropin  before they were mated. The fertilized eggs were flushed from the oviducts with pre-warmed HEPES-balanced RD medium , 2 mM/L HEPES, 2 mM/L L-glutamine and 100 μM/L NEAA), harvested in B2 medium prepared as previously reported2 and 95% air at 38 °C for 24 hr.The mRNA and ssODN mixture included 20 ng/μL Cas9 mRNA and 10 ng/μL sgRNA1F and sgRNA2R. Fifty millimolar ssODNs in the final concentrations were prepared immediately before micromanipulation. Approximately 5 pL of the mixture was injected into the cytoplasm of each zygote by a 3 µm diameter pipette using a Piezo instrument . After microinjection, zygotes were cultured in B2 medium in a humidified atmosphere containing 5% COCycles of female recipients were synchronized with zygote donors by injecting 100 IU hCG to induce ovulation. Approximately 20 embryos at the 4-cell or 8-cell stages were surgically transferred to both oviducts of each recipient through the infundibulum. Offspring were born naturally after 30 days of embryo transfer.Bln I  for analysis on a 2% TBE agarose gel. Furthermore, the PCR products were sequenced by Lifetech Co. .Genomic DNA was extracted from ear punch tissues of newborn kits by using a TIANgen Genomic DNA kit . The fragments were amplified from genomic DNA by PCR using the primers r78JDF and r78JDR. First, the fragments were analysed on a 1.5% TBE agarose gel for detection of gross deletion, and then the PCR fragments were used for T7EN1 cleavage assay. Second, the PCR fragments were purified by isopropanol and digested with The potential off-target sites of the two sgRNAs were predicted using the online-based tool Cas9. The top three POTS were selected for each sgRNA according to the ranking scores. The potential off-target sites were analysed by BLAST searching of the predicted position in Oryctolagus cuniculus (rabbit) nucleotide sequences. Approximately 400–600 bp genomic fragments containing the off-target sites were amplified by PCR and sequenced. The off-target sequence and PCR primers used in this study are shown in Supplementary Table Rabbit ceruloplasmin (CP) concentrations in plasma were quantitatively detected between the knock-out or knock-in rabbits and wild rabbits. Blood was drawn from the ear vein, and the plasma was separated by centrifugation at 4 °C, 4000 rpm. Then, the operation was executed according to the instructions accompanying a Rabbit CP ELISA Kit  on Spectramax i3 .Approximately 400 μL of plasma was treated by using a Copper Assay Kit according to the manufacturer’s instructions  and was analysed at a visible light wavelength of 580 nm with a biochemical analyser . Liver function indicators, including albumin (ALB), total protein (TP), alkaline phosphatase (ALP), glutamyl transferase (GGT), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were detected with the same machine using kits supplied by Ikon Biotechnology Co., Ltd. .ATP7B was mainly expressed in the liver and kidney. Therefore, 0.5 g of liver and kidney was extracted from the homologous mutation rabbits and the wild-type rabbits after euthanasia. All these reagents and protocols were implemented according to the instructions accompanying the Tissue Copper Colori Assay Kit . The tissues were cleaned and placed in a cryopreservation tube in liquid nitrogen overnight. The next day, the tissues were homogenized to obtain a supernatant for the detection of copper ion concentration at 580 nm using a spectrophotometer .The liver tissues of the rabbits were treated with 10% formalin for 24 hr, dehydrated in 75% ethanol for 24 hr followed by dehydration in different grades of alcohol, and vitrified with dimethylbenzene. After the tissues were embedded in paraffin, 5 μm longitudinal sections were stained with haematoxylin solution for 5 min and washed with running water and then stained with eosin solution for 20 sec. Finally, xylene was applied for clearing, and the samples were observed under a light microscope  after the samples were sealed with neutral balsam on slides.t-test. The level of statistical significance was set to P < 0.05.For all parameters, the parameters were test for three times, the average of three number and the error were show in the data, and expressed as means ± standard error. The mutation kits obtained by microinjection at different times were examined by a chi-square test to compare the KI frequencies obtained when zygotes were injected 19 hr after HCG treatment with the KI frequencies obtained when zygotes were injected 14 hr after HCG treatment. The significant differences in the biochemical index between muted and WT rabbits were analysed using Student’s Xba I digestion and transcription. The SgF0 fragment was amplified from the pCD-CAS plasmid  by PCR with primers and used as a template to amplify different target DNA fragments for transcription. After transcription and purification, two sgRNAs were obtained with sufficient concentration and quality according to biometric spectrophotometry.Cas9 mRNA was purified after The target fragments (541 bp) were cut by the Cas9 nuclease and combined with two sgRNAs. The Cas9 nuclease and sgRNA1F digested the fragment into two bands with lengths of nearly 240 bp and 300 bp, suggesting that the fragment was fully cut and that the efficiency was 100%. Three bands at 541 bp, 300 bp and 241 bp were detected after sgRNA2R digestion  pups with precision point mutations in the Arg780Leu locus and 7 (50%) KO pups. To improve the efficiency of point mutations, 56 fertilized eggs obtained at 14 hrs after HCG were injected with the mixture of RNAs and ssODNs. Consequently, 17 pups were obtained, of which 9 (52.94%) carried a point mutation and 5 (29.41%) were KO pups   Fig. . The resATP7B precision point mutations and KOs could be stably transmitted to offspring, adult mutant rabbits were used for reproduction. When the male rabbit F0–48 was mated with a wild-type (WT) female rabbit, two rabbits carrying the point mutation were derived from three offspring. When the male rabbit F0–24 was mated with the WT female rabbits, two rabbits carrying the KO were derived from four offspring  of which carried the off-target genotype. The rabbits that carried the off-target genotype were two of three rabbits in the offspring produced by mating F0–48 with a WT female rabbit. The genotype was also detected in three of seven rabbits in the offspring produced by F0–47 mated with F0–24. Eleven of these fourteen F1 offspring carried mutations, and six of them did not carry the off-target genotype.Ceruloplasmin is the major carrier of copper in the blood and is typically low in patients with WD. The ceruloplasmin level showed a downward trend in the mutant rabbits of the F0 Fig.  and F1  generatiThe plasma copper in patients with WD is usually decreased in proportion to the decreased ceruloplasmin in the circulation, whereas in patients with severe liver injury, plasma copper may be within the normal range despite the lower ceruloplasmin level. The results of plasma copper detection showed that most mutant rabbits had a lower plasma copper level than that of the WT rabbits in generation F0 Fig. . HoweverHepatocyte damage is one of the main features of WD. The following results were obtained when liver function indicators were measured in the F0 and F1 rabbits, and the above data were also compared between homozygous and WT individuals Fig. . The levATP7B mutant rabbits was 9 times higher than that in the wild rabbits (**P < 0.01). The copper in the kidney was 5 times higher than that in the wild rabbits (**P < 0. 01). In addition, the liver copper was higher than that in the kidneys of mutant rabbits (*P < 0.05)  and four WT litter-mate rabbit were detected for the accumulation of copper. The outcomes of tissue copper detection showed that the copper in the livers of the 05) Fig. .Figure 8HE staining demonstrated that liver of the mutant rabbits exhibited morphological changes relative to those of the wild-type rabbits. There was clear connective tissue proliferation in the livers of the mutant rabbits, and a hepatic pseudo-lobule appeared. Additionally, hepatic cell cords were not distinct Fig. .Figure 9ATP7B gene point mutation occur at the high homology site of the 780th amino acid arginine through homologous recombination. This disease model simulates the major mutation type in Asians with WD.This research was based on the use of micro-manipulation technology combined with an efficient and specific CRISPR/Cas9 system to make the ATP7B gene. The mutation of ATP7B may cause ceruloplasmin synthesis disorder and biliary copper excretion disorder, thus resulting in excessive copper deposition in various organs, particularly the liver and kidneys. The hotspot mutation in Europeans is in exon 14, whereas Arg778Leu in exon 8 in Chinese and Koreans is the first hotspot42. Protein-protein BLAST analysis indicated that the homology of the amino acid sequences between the human and the rat or mouse ATP7B proteins is only 50%  14 hours after injection of HCG was significantly higher than that 19 hours after injection of HCG (14.29%)(*p < 0.05) (Table CRISPR/Cas9 has been developed as an efficient genome engineering tool to introduce a site-directed point mutation with ssODNsin vitro cutting assay showed that sgRNA1F had a higher cutting efficiency than sgRNA2R; however, sgRNA2R has a lower off-target rate, as predicted online. Therefore, the two sgRNAs were used for all experiments. The results of DNA sequencing showed that most indel mutations occurred between the two PAMs of the couple sgRNAs. These two sgRNAs cutting may also facilitate precision point mutations for HDR repair using Cas9/gRNAs, and ssODNs utilize synthesis-dependent strand annealing pathways when double-strand lesions are derived by Cas945. When the ssODNs were annealed with the overhang broken strand end, the complementary strands were synthesized followed by flap cleavage, ligation and single-strand gap repair with another broken strand end. The sequence of another broken strand end may disturb the sequence of the ssODNs, and thus, only one part of the point mutation was introduced, e.g., only one base was replaced in three rabbits (Fig. In this study, two sgRNA were devised to cut the target sequence. The ATP7B point mutation and KO were stably transmitted to the offspring. Additionally, complex heterozygous and heterozygous rabbits were also produced from the mutant parents.Eight heterozygous rabbits grew into adults, and three of them were mated with each other or with wild-type rabbits. Genotype analysis of the F1 generation showed that the genotypes of the To address concerns about the possibility of the CRISPR/Cas9 system generating “off-target” changes to the rabbit genome, we analysed the genomes of all the founder rabbits by using a combination of predictive bioinformatics to identify possible off-target sites, PCR to allow for amplification and Sanger sequencing to identify off-target effects in these sites. The sgRNA1F produced off-target mutations—up to 60.87% of the highest-scoring-potential off-target sites—whereas the sgRNA2R did not. All fourteen F1 offspring were also detected for the off-target mutations, five of them carried both the target mutation and off-target mutation, and six of them carried the target mutation but not the off-target mutation; thus, the off-target mutation could be omitted in the offspring.ATP7B point mutation or knock-out rabbits exhibited defects and abnormalities in ceruloplasmin, copper in plasma, copper in tissues, morphology of liver tissue and indexes of correlation with liver function. These results suggest that mutation of ATP7B is insufficient for copper transport and metabolism in vivo, in agreement with findings from previous reports in humans47.In this study, we found that the 48. More significantly, we found a similar ratio of copper content in the liver between rabbits and humans at the onset of WD. There was a nine-fold increase in the mutant rabbits and a five-fold increase in humans compared with an approximately 30-fold increase in mice49.Ceruloplasmin is mainly synthesized in the liver and functions as a major carrier of copper in the blood. Using ceruloplasmin to identify patients with WD is further complicated by overlap with certain heterozygotes, whereas 20% of heterozygotes have decreased levels of ceruloplasmin. The ELISA results showed that the concentration of ceruloplasmin in KO and point mutant rabbits was lower than that in WT rabbits, in agreement with previous findings in humansThese rabbits died of WD in approximately three months. However, in humans, the onset of WD is at 5 to 50 years old. This significant difference may be attributed to two reasons: human WD is derived from different types of mutations and different WD patients exhibit varying levels of copper intake because they live in different regions and lead different lifestyles. All WD rabbits may have died early, before adolescence, because they carried the same mutation, had a similar genetic background, and lived on the same diet and because there is no cure for hepatitis. These features of the WD rabbit make it a potential research model for treatment testing. Therefore, more work should be conducted on this model in the future.50 and gene knock-in33 in rabbits. The present study developed a potential rabbit model for WD disease. Further validation of the model will be required to determine whether it is suitable for gene therapy research.In summary, this is a successful study to build a precision point mutation rabbit disease model using CRISPR/Cas9 technology following gene knockoutSupplementary Information


RUNX1 locus that fuse the RUNX1 DNA-binding domain to different regulators, the t expressing RUNX1-ETO and the t expressing RUNX1-EVI1. Despite containing the same DNA-binding domain, the two fusion proteins display distinct binding patterns, show differences in gene expression and chromatin landscape, and are dependent on different transcription factors. RUNX1-EVI1 directs a stem cell-like transcriptional network reliant on GATA2, whereas that of RUNX1-ETO-expressing cells is more mature and depends on RUNX1. However, both types of AML are dependent on the continuous expression of the fusion proteins. Our data provide a molecular explanation for the differences in clinical prognosis for these types of AML.Acute myeloid leukemia (AML) is a heterogeneous disease caused by mutations in transcriptional regulator genes, but how different mutant regulators shape the chromatin landscape is unclear. Here, we compared the transcriptional networks of two types of AML with chromosomal translocations of the   •t and t AML are regulated by unique gene regulatory networks•RUNX1-ETO and RUNX1-EVI1 have different binding patterns and binding partners•Depletion of each fusion protein initiates C/EBPα-dependent myeloid differentiation•t AML survival depends on RUNX1, whereas t AML survival requires GATA2 Loke et al. compare the regulatory signature of two related types of acute myeloid leukemia, t expressing RUNX1-ETO and t expressing RUNX1-EVI1. Each fusion protein displays a distinct binding pattern and cooperates with different transcription factors to impact the epigenome, but both downregulate the myeloid differentiation regulator C/EBPα. Acute myeloid leukemia (AML) is the most common acute leukemia in adults. Despite improvements in supportive care, outcome typically remains poor for AML patients older than 60 years who are unfit for intensive chemotherapy . AML is Mutations involving the hematopoietic master regulator RUNX1 are among the most commonly found abnormalities in AML. RUNX1 is the DNA-binding component of core binding factor (CBF), binding as a dimer with CBFβ, which is encoded by another recurrently rearranged gene in AML. The most common category of RUNX1 rearrangement is the product of the t chromosomal translocation, RUNX1-ETO, which comprises the RUNX1 DNA-binding domain linked to the almost complete ETO protein , which functions as a repressor by recruiting histone deacetylases  Figure A. The t. RUNX1-ERUNX1 translocation, t, is RUNX1-EVI1, whereby the RUNT domain is fused to the entire EVI1 gene  encodes a dual domain zinc-finger transcription factor with direct DNA-binding activity together with a histone methyl transferase (SET) domain (The product of another VI1 gene A (MitaniVI1 gene . EVI1 (a) domain   domain A and is ) domain . The t(3) domain  and is m) domain  or as a ) domain .RUNX1-EVI1 knocked into the RUNX1 locus display a phenotype similar to the RUNX1-ETO knockin  and two t AML patients, two sets of normal CD34+ progenitor cells purified as mobilized peripheral blood stem cells (PBSCs) from peripheral blood, and a t cell line derived from a CML patient in blast crisis  malignancies, but not in t AML (+ cells (PBSCs) than t cells  and t AML we mapped the accessible chromatin landscape by identifying all DHSs in purified CD34s SKH-1; ). We pers SKH-1; , and ana;21) AML . Correla;21) AML C and 1D 1) cells  C and 1D and t cells G (Ptasin1) cells . In cont+ cells  and the PU.1 gene (SPI1), the two fusion proteins target the same regulatory elements  than in t  cells have shown that RUNX1-ETO co-associates with a number of hematopoietic regulators such as the E-box-binding protein HEB; the ETS factors ERG, FLI1, and PU.1; and the LMO2/LDB1 complex . To test t C. Other  t . ChIP ex t D, which  t C and S3E t D and cel t D. Such aTo validate our ChIP data in primary cells, we performed digital DNase I footprinting and identified regions protected from nuclease digestion indicative of transcription factor binding using the Wellington algorithm . We thenOur binding data suggested that RUNX1-EVI1 and RUNX1-ETO associate with different transcription factor complexes. Furthermore, such differential binding is also found with the wild-type RUNX1 protein expressed from the non-translocated allele in each AML type, indicating that RUNX1 fulfills different roles in programming the chromatin landscape in each cellular context. It was previously shown that the survival of t cells is dependent on the expression of wild-type RUNX1, whereby RUNX1 regulated a complementary set of genes balancing the effects of RUNX1-ETO . We therPrevious studies have shown that the members of the RUNX1-ETO complex (namely LMO2 and ERG) are required for the leukemogenicity of RUNX1-ETO and their survival . To gainTaken together, our data demonstrate that despite sharing the same DNA-binding domain, the two fusion proteins predominantly bind to different genomic sites, co-bind with different partners, and operate within a different chromatin landscape. Moreover, the transcriptional networks regulating the survival of both types of AML depend on different non-mutated transcription factors, with t cell depending on RUNX1 and t cell being dependent on GATA2.GATA2  translocation is a driver mutation , and RUNGATA2 A–5C, whend CEBPA F. The exaffected D. Gene saffected A and S5Band JAK3 I. A para and MPO F. A more and MPO J. This aGATA2 and the upregulation of CEBPA expression after RUNX1-EVI1 knockdown  cells after RUNX1-EVI1 knockdown, we examined the changes in the epigenetic landscape of t SKH-1 cells by mapping DHSs in cells treated with a control siRNA or after 10 days of knockdown with a RUNX1-EVI1-specific siRNA C. Examplnockdown A and 5H.nockdown C and didnockdown E and S6Bnockdown D and S6CTA2 gene A. These TA2 gene D and 6E TA2 gene D. An aliTA2 gene F. In conTA2 gene F. CEBPA PA locus E.Figure HOXA9 after RUNX1-EVI1 knockdown, slowed the decrease of the key stem cell renewal genes GATA2 and MEIS1, and blocked increased expression of the markers of myeloid differentiation CTSG, MPO, and CSF1R  to block C/EBPα binding  during knockdown RUNX1-EVI1 by siRNA. The DNCEBP peptide dimerizes with CEBP transcription factors and prevents binding to DNA . Expressnd CSF1R B. ChIP end CSF1R F. Anothend CSF1R F and 7G tivation H and S7DTaken together, as summarized in The study presented here used global analyses to investigate differences and similarities between two types of CBF AML: the t expressing RUNX1-ETO and the t expressing RUNX1-EVI1, which both carry the same RUNX1 DNA-binding domain. Our DHS mapping, digital footprinting experiments, and ChIP assays of patient cells and appropriate patient-derived model cell lines unequivocally determined how (1) the epigenetic and transcriptional profiles of the two types of AML differ and (2) show that the RUNT DNA-binding domain of each fusion protein is not the sole determining factor for the selection of fusion protein-binding sites in the genome. Moreover, each type of AML displays a unique, stable transcriptional network that is dependent on the presence of each fusion protein but requires a different set of associated transcription factors.Spi1(PU.1) and Cebpa and a concomitant increase in the expression of stem cell genes such as Gata2 and Erg, indicating extensive feed-forward loops driving myelopoiesis  regulating leukemia aggressiveness , but not in t, AML. HOXA9 expression is associated with poor prognosis  as compared to t AML. RUNX1-EVI1 appears to directly regulate a stem cell program establishing an immature phenotype associated with treatment resistance, , expresssiveness . Furtherrognosis , and is rognosis . MEIS1 eand LSCs . HOXA9 ad in AML  and Hoxaor cells . This coor cells .ERG in AML is generally associated with poor prognosis  cells than in t cells, which can be explained by a differential activity of its cis-regulatory elements. Our data show that a distal GATA2 enhancer, known to upregulate GATA2 expression  patient cells, but neither RUNX1 nor RUNX1-EVI1 binds to this element in t cells.The different gene regulatory networks maintaining the two types of AML involve alternate sets of transcription factors, and they differentially program the chromatin landscape, thus impacting where the fusion proteins bind. Our data show that RUNX1-ETO-binding sites are enriched for occupied ETS/RUNX/E-box motifs, reflecting the structure of the RUNX1-ETO complex, with the ETS factors ERG and FLI1 in the complex being required for leukemia maintenance and leukemogenesis . The exprognosis . In contrognosis , but notrognosis , which mpression , is accecis-regulatory modules, which through auto-regulation of genes encoding complex members initiate the formation of stable gene regulatory networks that ultimately define the behavior of each type of AML . This result complements previous data showing that overexpression of CEBPA can overcome the RUNX1-EVI1-mediated differentiation block  and t transcriptional networks, C/EBPα is downregulated in both types of AML, suggesting that it is a critical node by which leukemia is maintained. We show that C/EBPα is directly repressed by both RUNX1-EVI1 and RUNX1-ETO through binding to a recently characterized upstream enhancer . In bothnockdown  or expreon block . An inteon block . This abTaken together, our study provides an important paradigm for studies aimed at understanding how different leukemic fusion proteins program and interact with the epigenetic landscape in two related but different types of AML. Our data represent a resource that will facilitate global mechanistic studies of the genes, transcription factors, and pathways involved in blocking myeloid differentiation and emphasize that different types of AML, despite being a disease of one specific differentiation pathway, are maintained by highly diverse transcriptional networks. Our study therefore highlights the complexities we have to face in our understanding of AML heterogeneity if we want to use this knowledge to devise AML-specific therapies.Cells were purified as described previously , with mi7 cells were electroporated using an EPI 3500 (Fischer) at 350 V, 10 ms. siRNA sequences are listed in the 6 cells/mL, returned to an incubator, and kept at 37°C and 5% CO2.1 × 10DHS mapping, ChIP-seq, and digital footprinting using the Wellington algorithm  was perfDetails of data analyses can be found in All human tissue was obtained with the required ethical approval from the National Health Service (NHS) National Research Ethics Committee. Detailed information about patient samples is listed in J.L. performed experiments and wrote the paper. M.R.I., A.P., Y.G., and N.M.S. performed experiments. S.A.A. and P.C. analyzed the data. M.R. and P.N.C. helped supervising the study and writing the paper. H.R.D. provided essential research material. O.H. designed and supervised experiments and helped writing the paper. C.B. designed the study, supervised experiments, and wrote the paper.


In The Cancer Genome Atlas (TCGA) CN-AML patients, higher scores were found to be associated with both worse OS  and DFS . Results of gene ontology (GO) analysis showed that all the significant GO Terms were correlated with cellular component of mitochondrion. In summary, a novel gene set that could predict prognosis of CN-AML was identified presently, which provided a new way to identify genes impacting AML chemo-sensitivity and prognosis.The prognosis of cytogenetically normal acute myeloid leukemia (CN-AML) varies greatly among patients. Achievement of complete remission (CR) after chemotherapy is indispensable for a better prognosis. To develop a gene signature predicting overall survival (OS) in CN-AML, we performed data mining procedure based on whole genome expression data of both blood cancer cell lines and AML patients from open access database. A gene expression signature including 42 probes was derived. These probes were significantly associated with both cytarabine half maximal inhibitory concentration values in blood cancer cell lines and OS in CN-AML patients. By using cox regression analysis and linear regression analysis, a chemo-sensitive score calculated algorithm based on mRNA expression levels of the 42 probes was established. The scores were associated with OS in both the training sample ( Acute myeloid leukemia (AML), characterized by the rapid growth of abnormal white blood cells interfering with the production of normal blood cells, is the most common type of acute leukemia affecting adults. Presently, induction chemotherapy with cytarabine and anthracyclines is the first-line treatment for AML except for acute promyelocytic leukemia . However, outcomes of AML vary greatly among patients after chemotherapy. Clinical studies have shown that the five-year survival rate of AML varies from 18% to 82%, and relapse rate varies from 33% to 80% .NPM1, FLT3-ITD, CEBPA, WT1, ASXL1, IDH1/2, DNMT3A, and RUNX1 [LFE1 [CXXC5 [EVL1, MEL1 [miR-9* [It is well known that chromosomal abnormalities are major prognostic factors in AML. Based on karyotype, AML patients can be divided into favorable, intermediate and unfavorable cytogenetics risk groups which show different survival profiles . Howevernd RUNX1 –10. MoreX1 [LFE1 , CXXC5 [1 [CXXC5 , EVL1, ML1, MEL1 , and miR [miR-9*  are alsoin vitro. Previous studies have confirmed that the use of pharmacological data and genomic information of cancer cell lines can help researchers to identify therapeutic biomarkers [Cancer cell lines, which were descended from naturally occurring tumors, are commonly used for molecular biology research and drug discovery omarkers –17. Thesomarkers –20. Theret al. reported an 86-probe-set mRNA expression signature which was correlated with overall survival (OS) of AML [et al. found a leukemic stem cell gene expression signature correlated with clinical outcomes in AML [et al. derived a lncRNA score composed of 48 lncRNAs and found that the score was an independent marker for the outcome in CN-AML patients [50) values for 138 anticancer drugs on more than 800 cancer cell lines. Whole genome mutations and expression levels are also available for the GDSC database. To find out whether genes correlated with chemo-sensitivity could also predict the outcome of AML, we downloaded IC50 values of cytarabine from 96 leukemia cell lines and raw whole genome expression data of these cell lines from GDSC in this study. Probes significantly associated with IC50 levels in the cells and OS in GSE12417 dataset, a whole genome expression dataset for CN-AML in NCBI Gene Expression Omnibus (GEO), were selected. Based on the selected probes, chemo-sensitivity score was derived and replicated in a TCGA CN-AML dataset.Recently, a lot of classified studies based on RNA microarray have been performed to establish the classifier which might help to predict the outcomes of CN-AML. For example, Metzeler ) of AML . Gentless in AML . Garzon patients . Howeverpatients . This da50 values between the two classes was observed  and U-698-M (a B-Acute Lymphoblastic Leukemia cell line) Figure . Signifi50 values (raw p<0.05) in the GDSC blood cell lines. GO annotation was carried out to predict the potential function of genes targeted by the probes. In terms of biological processes, 4 of the top 10 categories belonged to RNA processing modification and metabolic process . Finally, 42 probes correlated with both cytarabine sensitivity and OS in the same direction were identified. Details of these probes were listed in Table p<0.05 were identified, and four  of them passed correction for multiple tests .The detailed information including univariate cox score and combined cox score of the 42 selected probes mapped to 44 annotated genes were summarized in Table de novo CN-AML patients was also assessed. Patients with an unfavorable score had a shorter OS . As disease-free survival (DFS) data was also available for the TCGA AML dataset. Our results showed that patients with an unfavorable score had significantly shorter DFS as compared with patients with a favorable chemo-sensitivity , used for the treatment of acute promyelocytic leukemia (APL), could induce apoptosis at therapeutic concentrations (1–2mM) through the mitochondrial pathway in APL NB4 cells [For the 44 genes involved in the signature, results of pathway analysis by using the David database showed that 4 GO terms were significantly correlated with cellular component of mitochondrion after multiple testing corrections. Mitochondria is a key organelles in human cells participating in cell apoptosis , 29, andhenotype . Xiao ete in AML . Wang etB4 cells .There is also study shows that AML blast cells have higher copy number of mtDNA and consume more oxygen . When AMSLC14A1 was found to be upregulated in cytarabine-resistant cells [SLC14A1 is also included in our gene signature, but in an opposite direction. In our results, SLC14A1 high expression correlated with low IC50 values in blood cancer cell lines and acted as a good prognostic predictor in AML patients for both GSE12417 and TCGA CN-AML datasets. We further queried this gene in GEO database, and found that there were remarkable inter-individual variations in the expression of SLC14A1 in AML patients  study that focused on cytarabine sensitive and resistant murine cell lines, nt cells . Interespatients . In the patients . This maleukemia . As in o results . Moreovell lines . We alsorst time , 44, 45.50 of cytarabine. Therefore, this process might cause false positive results due to specificity of blood cancers. Finally, we failed to validate our prediction model in prospective study. Therefore, further studies are required to validate the clinic significance of the model.There are also some limitations for this study. First of all, the data used in this study is obtained from public database, therefore information for some common AML somatic mutations and other known factors affecting AML drug response were not available for constructing prediction model, which may result in uncertainty of whether the chemo-sensitivity score was an independent factor. Integration of gene expression data and somatic mutation data is supposed to build a more precision prediction model. Secondly, only 17 AML cell lines are deposited in GDSC database, and in order to gain better statistic power of linear regression analysis, all the blood cancer cell lines were used to screen the probes that were associated with ICIn conclusion, we identified a novel gene set that could affect both the cytarabine sensitivity in blood cell lines and OS of AML patients underwent cytarabine therapy. The prediction model based on the gene set could predict prognosis of CN-AML. Most genes included in the set are correlated with mitochondrial, which hints that mitochondrial might be important in cytarabine response and AML outcomes. Our study proposed a new way to identify factors impacting AML prognosis, and this would provide some scientific basis for elucidating the individual difference in outcomes of AML and realizing personalized medicine according to gene expression pattern of AML.http://www.cancerrxgene.org/) includes more than 800 cancer cell lines with whole genome mRNA expression, mutation and copy number variation information. Based on GDSC release 5.0, 96 blood cancer cell lines with both natural log transformed IC50 values of cytarabine and normalized whole genome mRNA expression levels were selected in our study  was conducted to confirm whether the probes affect OS of CN-AML patients. Probes exhibited significance (p<0.05) in both the linear-regression analysis and the cox regression analysis in the same direction were screened. For example, if high expression of a probe was associated with high cytarabine IC50 value, the high expression of this probe must correlate with poor AML prognosis.Linear-regression analysis was performed to identify probes correlated with cytarabine ICCCSi=αi1×β1+αi2×β2, where CCSi was the combined cox score of probe i. Because GSE12417 included two samples, αi1 and αi2 represented the univariate cox score for U133 AB sample and U133 plus sample, respectively; while β1 and β2 were the proportion of U133 AB and U133 plus samples in GSE12417, respectively. To get the chemo-sensitivity score of each sample, the following formula was used: CSj was the chemo-sensitivity score of patient j, n was the number of selected probes, and γij was the expression level of probe i in patient j.To obtain the chemo-sensitivity score for each patients, the expression values of the selected probes were standardized transformed . Then, the univariate cox scores (which indicated the correlation between probe expression levels and OS of the AML patients in GSE12417) of the selected probes were acquired and used to calculate combined cox score for each probe by the following formula: https://david.ncifcrf.gov/). The GO analysis was performed based on biological process (BP), cellular component (CC) and molecular function (MF). The Kaplan-Meier plots were generated by Graphpad 5.0. All of the data arrangement and statistical analysis were conducted by using R (version 3.1.2). Methods flow chart of analysis process was shown in Figure To obtain the Kaplan-Meier plots of chemo-sensitivity score, patients were divided into favorable and unfavorable groups based on their chemo-sensitivity scores. The median was used as cut-point. Patients with chemo-sensitivity scores equal to or higher than the median were classified as unfavorable, the others were classified as favorable. Heatmap.2 command in gplots package was utilized for the cluster analysis. Gene Ontology (GO) and pathway analysis were conducted by the tool David 6.7 (


In contrast to wild lagomorphs, pet rabbits exhibit a noticeably high frequency of dental problems. Although dietary habits are considered as a major factor contributing to acquired malocclusions, the exact causes and interrelationships are still under debate. In this regard, an important aspect that has not been considered thoroughly to date is the effect of diet-induced phenotypic plasticity in skull morphology. Therefore, we conducted a geometric morphometric analysis on skull radiological images of wild and pet rabbits in order to quantify intraspecific variation in craniomandibular morphology. The statistical analyses reveal a significant morphological differentiation of the craniomandibular system between both groups. Furthermore, the analysis of covariance shows that the force-generating modules (cranium and mandible) vary independently from the force-receiving module (hypselodont teeth) in pet rabbits, which is in contrast to their wild relatives. Our findings suggest that the phenotypic changes in domestic rabbits impact mastication performance and, consequently, oral health. An adequate close-to-nature nutrition throughout the whole life and especially beginning early parallel to weaning (phase of increased phenotypic plasticity) is necessary to ensure a normal strain on the teeth by promoting physiological lateral gliding movements and avoiding direct axial loads. Oryctolagus cuniculus, which is a member of the family Leporidae (rabbits and hares). A remarkable peculiarity in veterinary medicine is the prevalence of dental problems among small herbivorous pet animals in general and rabbits in particular rticular ,7,8,9. Srticular ,4,10,11.O. cuniculus. Yet, the covariation among distinct modules in the skull of rabbits and the link between morphological variation and functional implications (in particular mastication) remains largely unknown.It is well known that the evolution of wild species into domestic forms by artificial selection has resulted in changes in behavior and morphology ,13 and vO. cuniculus in order to understand the significant role of the highly specialized masticatory apparatus in rabbits including their hypselodont teeth.A major factor contributing to the alarming situation with regard to dental problems in pet rabbits is the feeding behavior ,32, whicOryctolagus is noted in the fossil record, associated with an arid, warm savannah-type fauna [Oryctolagus persists until today, but with only one species (O. cuniculus) left, and the last glacial period confined it to the Iberian Peninsula and southern France [The evolutionary history of Leporidae dates back to the Eocene  and is lpe fauna . The glope fauna . Oryctoln France . About 1n France ,41 and hn France .Due to the interaction between climate and vegetation, the global climate change in the early Cenozoic affected the evolution of the herbivorous rabbits. In this context, a main factor limiting the life span of mammals in the wild is tooth abrasion e.g., . HypseloO. cuniculus in order to address the following questions: (1) how does the entire skull morphology vary between wild and domestic rabbits; (2) to what extent is morphological variation in the skull modular and (3) what are the implications for the masticatory apparatus of domestic rabbits. Ultimately, we seek to provide an explanation for the high abundance of dental problems in pet rabbits from an evolutionary perspective. This will not only improve our understanding of the relation between morphology and pathologies in domestic animals, but is also an important case study of Evolutionary Veterinary Science (EvoVetSci).Despite its importance for veterinary medicine, it is surprising to note that we still lack knowledge about the patterns of morphological changes in the skull and dentition of a number of domestic animals including rabbits. Here, we quantify skull morphology in O. cuniculus. The wild rabbits originate from southern Germany and Austria (victims of traffic accidents). The sex is unknown. Due to the aforementioned prevalence of dental problems among domestic rabbits , the sample is somewhat limited, but sufficient to yield a reasonable signal. The pet rabbit sample represents no specific breed with erect ears in order to avoid extreme phenotypes characteristic of certain breeds. The specimens are radiographed for medical reasons and not for the purpose of this study. Only specimens with adult dentition and without pathologies are included. Skull radiographs  of anesthetized specimens are obtained with the mouth closed or open about one millimeter [In this case study, we examine the skulls of 12 mature European wild rabbits and 12 mature domestic rabbits belonging to the species llimeter .Landmark-based geometric morphometrics was used to quantify skull morphology and to analyze phenotypic differences. A total of 14 landmarks (LMs) in two dimensions were taken on laterolateral radiological images of the skull C. The hoAll subsequent analyses and statistical tests were performed using the software PAST . In ordeModular covariation of the shape between cranium, mandible and cheek teeth is investigated using two-block partial least squares (2-block PLS) analysis  in the sThe collected 2D LM coordinates of the analyzed specimens are available in About 95% of the total variance in the sample is explained by the first nine RWs . The firp-value < 0.001), predicting major differences between both groups. t-test: p-value > 0.05).The Hotelling’s T² test on the first nine RWs reveals that the mean difference values are significantly high . Means of log centroid size do not differ significantly between both groups (p > 0.05). Thus, the skull of pet rabbits reveals a larger variation in size than their wild counterparts. However, the average size is similar in both groups.The variance of log centroid size in wild and domestic rabbits is significantly different (p < 0.05). The amount of shape variation accounted for by the regressions is about 16.88% and 51.10%, respectively (Allometry of the skull accounts partly for a portion of shape variation among rabbits. The multivariate regression of the shape variables (RW 1–9) on log centroid size shows that shape variation associated with RW 1, 3 and 5 to 9 are not a function of size . There iectively . The ploectively . The latectively .Comparing the consensus shape (mean shape of the sample) with all landmark configurations after Procrustes superimposition visualizes the shape variation within the sample B. It indRW 2 and to a lesser extent RW 1 separate wild and domestic rabbits. RW 1 and RW 2 primarily contrast anteroposteriorly elongated and dorsoventrally compressed skulls  against those that are anteroposteriorly compressed and dorsoventrally expanded  C. This pFocusing on RW 2, skull shape changes associated with positive RW 2 values include a shortening of the occipital region in anterior direction and a slight shift of the anterior cranial region  in dorsal direction C. NegatiPLS 1 explains between 60% and 90% of the covariance between the three modules . TestingEvolution of craniomandibular shape in rabbits has been governed by ecological adaptation  includinIn accordance with the breeding for “cuteness” , the present analysis reveals that the skull shape is generally more quadratic in domestic rabbits, whereas wild rabbits tend to have a long and flat skull. In particular, the relative length of the nasal bone (represented by LM 1) and the occiput (represented by LM 14) characterize this difference. In domestic rabbits, the reference line that marks the dorsal limitation of the maxillary tooth apices in lateral view of the skull is defined to connect the most anterior point of the nasal bone with the most posterior point of the occipital protuberance  or the evolution towards a shorter skull. This is based on the fact that despite the significant difference in the shape of the cranium between wild and domestic rabbits, the morphological configuration of the teeth themselves (represented by LM 2–11) is very similar across all samples. However, a closer look at the three sub-units of the dentition  reveals that the tip of the maxillary incisors tends to project more ventrally in relation to the tip of the mandibular incisors in wild rabbits. This indicates slightly longer clinical crowns of the maxillary incisors. In contrast, the maxillary and mandibular incisors in domestic rabbits tend to occlude more bluntly with a slightly less chisel-shaped tooth tip. This may increase predisposition to incisor malocclusions with subsequent cheek tooth overgrowth.Normally, incisors and cheek teeth are kept in shape by the continual processes of attrition and abrasion, respectively, which are compensated by continuous growth of the teeth (hypselodont teeth). Accordingly, in rabbits with a healthy dentition, an eruption rate of approximately 2.0 mm per week was recorded in the maxillary incisors and 2.4 mm per week in the mandibular incisors ,61. The In rabbits with a healthy dentition, the incisors are continuously worn down during the biting and chewing of each masticatory cycle . While rIn addition to that, incisors are worn down continuously while pieces of food are ground between the cheek teeth, provided the food is suitable for physiological jaw movements with a rostrolingually oriented shearing power stroke. This kind of jaw movement is most pronounced in hay mastication and causes that the tips of the mandibular incisors move forward till they touch the dorsal edge of the wear facet of the maxillary incisors (circular upward motion). Furthermore, they are swept transversely across the caudal aspect of the second maxillary incisors . HoweverThe present landmark analysis shows that the area lying between the maxillary and mandibular diastema  is relatively long and flat in wild rabbits, whereas it is distinctly shorter and higher in domestic rabbits. In pet rabbits with a healthy dentition, the reference lines that mark the inclination of the palatine and mandibular bone plate slightly converge rostrally . The area which is defined by the antegonial notch of the mandibular ramus, the angular process and the posterior intersection between mandible and last mandibular molar (m3)  forms a nearly right-angled triangle both in wild and domestic rabbits. In the latter, however, the mandibular ramus is posteriorly higher according to the relative position of the angular process which is gently shifted dorsally C. This rIn this context, it is interesting to note that the part of the mandible that lies ventrocaudal to the antegonial notch  is more pronounced in domestic rabbits than in their wild counterparts A. This mIt is generally assumed that in rabbits, dietary habits seem to be a major factor in developing acquired malocclusions (reviewed in ). AlthouEach masticatory cycle consists of a biting (see above) and chewing sequence . ChewingDuring the shearing stroke which is primarily used in hay mastication the working side condyle moves forward from a strongly retracted position while the balancing condyle shifts slightly backward. Consequently, the mandibular cheek teeth on the working side are moved lingually (3–4 mm) and slightly rostrally (1 mm) with minimum vertical and maximum transverse jaw excursion . Thus, tThis jaw movement sometimes occurs in pellet mastication, but is never seen in carrot mastication. Carrots are always chewed with the aid of a crushing stroke where the position of the working condyle is initially more anterior. The forward movement of this condyle is less pronounced while the backward movement of the balancing condyle is enhanced. The mandibular cheek teeth of the working side move purely lingually without a rostromedial shearing action, just swinging slightly upward in the buccal and swinging slightly downward in the lingual phase. There is a maximum vertical gape and the result is primarily a crushing action that can also be observed in rabbits eating pellets. However, this type of chewing is never seen in hay mastication.In summary, this suggests that in grazing wild rabbits cheek teeth are strained primarily in a laterorostral direction while shear forces on the interlocked enamel crests dominate and there is only a small amount of axial load on the cheek teeth. In this context, it is reasonable that the first mandibular cheek tooth is the largest of the rabbit dentition. Thus, the teeth lying behind it can firmly prop up against this stronger premolar. In contrast to wild animals, most pet and breeding rabbits predominantly crush “unnatural” food between their teeth  which is accompanied with a much higher axial strain on the (pre-)molars and an insufficient tooth wear  combined with a tendency to retrograde tooth elongation . This faConsidering additionally that hay is more resistant than fresh grasses, it seems logical to develop further the hypothesis that pet and breeding rabbits had to develope stronger jaw muscles and secondarily larger axial bite forces than their wild counterparts to be able to crush their unnatural food more effectively. This might be supported by a shorter skull and more vertically oriented muscle fibers whereas a longer skull with a more anteriorly positioned masseter muscle (as seen in wild rabbits) reduces the vertical bite force due to a greater distribution of bite forces on all cheek teeth. As teeth at the rear of the dentition generally exert higher bite forces than the more rostrally positioned teeth, this might be an explanation for the found tendency of the cheek teeth to shift caudally in the group of the domestic rabbits. Furthermore, the presence of stronger muscles may explain the more salient appearance of the caudoventral part of the masseteric fossa (mandibular angle) in pet rabbits, as in different mammals (re-)modeling of the mandibular cortical bone has proven to be associated with oral processing of tough food (reviewed in ). This rA series of studies have supported the hypothesis that an increase in jaw robustness is an evolutionary or plastic response (phenotypic plasticity) to generating higher-magnitude loads. They all found load-related morphometric variations and phenotypic changes in jaw and skull morphology in many different mammals  being fed diets of different mechanical properties ,97,98,99All studies showed a positive correlation between dietary properties and peak masticatory loads that caused the adjacent cortical bony tissue to change its structure and morphology whereby it normally became thicker and more mineralized. Therefore, rabbits and primates that routinely ingested stiff and tough food exhibited relatively larger jaws to counter elevated peak masticatory stresses (peak bite force) (reviewed in ). In rabThe present study is an intriguing example that highlights the importance of integrating evolution and veterinary science in order to improve the knowledge base. Evolutionary Veterinary Science is key to gain a comprehensive understanding of pathologies and, thus, opens up new avenues of research in veterinary medicine.In summary, the landmark-based geometric morphometric analysis indicates that the craniomandibular shape of rabbits changed at different rates in the course of domestication since cranial morphometry strongly differs between domestic and wild rabbits although the dentition itself does not seem to differ significantly. This leads to a functional imbalance of the masticatory apparatus because the regions that are associated with the generation of masticatory forces  change independently from the regions that are associated with the resistance of masticatory forces . Finally, this disequilibrium seems to result in a predisposition to dental problems in domestic rabbits. What caused shorter skulls in the course of the domestication? On the one hand, selective breeding for extremely short crania in dwarf rabbits is sometimes accompanied with the occurrence of extremely short skulls . On the other hand, it has been proven that diet has a significant influence on skull morphology as well (phenotypic plasticity) .The present analysis comparing wild and domestic rabbits shows that in pet animals an increased skull height with a concurrently greater muscle insertion area  and more vertically oriented jaw muscle fibers exhibit higher muscle strength and, thus, a larger bite force compared to wild rabbits. Previous studies confirm that the shorter skull morphology seems to be a long-term adaptation to the increased stress on the dentition due to feeding a diet consisting predominantly of harder particles than that found in the wild  . Instead