Knock-in of exogenous sequences based on CRISPR/Cas9 targeting autosomal genes and sex chromosomes in the diamondback moth,Plutella xylostella

Genetic pest control strategies based on precise sex separation and only releasing sterile males can be accomplished by site-specific genome editing.In the current study,we showed that the mutation of single-allele Pxfl(2)d can significantly impair the normal mating behavior and testis development in male adults of the notorious cruciferous insect pest Plutella xylostella,in addition to its known functions in the ovarian development in female adults and egg hatching.Subsequent CRISPR/Cas9-based knock-in experiments revealed that site-specific integration of an exogenous green fluorescent protein(GFP)gene into autosomal Pxfl(2)d for labelling mutants could be achieved.However,this gene is not a suitable target for GFP insertion to establish a genetically stable knock-in strain because of the severe decline in reproductive capacity.We further screened for the W-chromosome-linked and Z-chromosome-linked regions to test the knock-in efficiency mediated by CRISPR/Cas9.The results verified that both types of chromosomes can be targeted for the site-specific insertion of exogenous sequences.We ultimately obtained a homozygous knock-in strain with the integration of both Cas9 and cyan fluorescent protein(CFP)expression cassettes on a Z-linked region in P.xylostella,which can also be used for early sex detection.By injecting the sgRNA targeting Pxfl(2)d alone into the eggs laid by female adults of the Z-Cas9-CFP strain,the gene editing efficiency reached 29.73%,confirming the success of expressing a functional Cas9 gene.Taken together,we demonstrated the feasibility of the knock-in of an exogenous gene to different genomic regions in P.xylostella,while the establishment of a heritable strain required the positioning of appropriate sites.This study provides an important working basis and technical support for further developing genetic strategies for insect pest control.

knock-in法建立Cre重组酶表达鼠系

目的 建立神经特异性表达的Cre重组酶鼠系 ,以便在特定的组织中 ,于某一特定的时间段 ,用条件性基因剔除技术在活体状态下研究基因功能。方法 用knock -in技术 ,以微小管相关蛋白 1a(Mapla)启动子控制Cre重组酶表达。结果 PCR检测结果表明 ,Cre重组酶基因已成功植入该鼠系中。结论 本研究为配合条件性基因剔除技术 。

Spectrum-effect relationship of immunologic activity of Ganoderma lucidum by UPLC-MS/MS and component knock-out method

This study was designed to elucidate the immunoregulation of Ganoderma lucidum.HPLC fingerprint and spectrum-effect relationship of G.lucidum were established to predict the active compounds and BP neural network model was established to predict the efficacy.Then the target compounds were identified by high resolution mass spectrometry.The results indicated that there are both enhanced immunity and immunosuppressive components in G.lucidum.BP neural network was trained with the common peak area and immune effi cacy index of G.lucidum fi ngerprint as samples,and a combined evaluation system of G.lucidum fi ngerprint effi cacy was established.The correlation coeffi cient R of BP network model was 0.98643,and the error of pharmacodynamic prediction results was in the ideal range.Eight compounds were identifi ed by high resolution mass spectrometry.The compounds related to immune activity in G.lucidum were determined in this study.

Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat（CRISPR）/Cas9-mediated gene knock-ins with zinc finger nucleases（ZFNs） and transcription activator-like effector nucleases（TALENs） in bovine and dairy goat fetal fibroblasts. To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin（MSTN） gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2. Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in theGFP-PGK-Neo R plasmid background, including a 5′ and 3′ homologous arm flanking the genes humanized Fat-1（h Fat-1） or enhanced green fluorescent protein（eGFP）. Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and thehFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts. After G418（Geneticin） selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove. The gene knock-in events were screened by PCR across the homologous arms. The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP andhFat-1 gene knock-ins were 13.68 and 0%, respectively. The efficiencies of CRISPR/Cas9-mediated eGFP andhFat-1 gene knock-ins were 77.02 and 79.01%, respectively. The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system. Additionally, thehFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system. The difference of knockin efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP andhFat-1 gene knock-ins were 32.35 and 26.47%, respectively. Theefficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively. The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs. The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines.

Studies on the temporal,structural,and interacting features of the clubroot resistance gene Rcr1 using CRISPR/Cas9-based systems

Clubroot disease is a severe threat to Brassica crops globally,particularly in western Canada.Genetic resistance,achieved through pyramiding clubroot resistance(CR)genes with different modes of action,is the most important strategy for managing the disease.However,studies on the CR gene functions are quite limited.In this study,we have conducted investigations into the temporal,structural,and interacting features of a newly cloned CR gene,Rcr1,using CRISPR/Cas9 technology.For temporal functionality,we developed a novel CRISPR/Cas9-based binary vector,pHHIGR-Hsp18.2,to deliver Rcr1 into a susceptible canola line(DH12075)and observed that early expression of Rcr1 is critical for conferring resistance.For structural functionality,several independent mutations in specific domains of Rcr1 resulted in loss-offunction,highlighting their importance for CR phenotype.In the study of the interacting features of Rcr1,a cysteine protease gene and its homologous allele in canola were successfully disrupted via CRISPR/Cas9 as an interacting component with Rcr1 protein,resulting in the conversion from clubroot resistant to susceptible in plants carrying intact Rcr1.These results indicated an indispensable role of these two cysteine proteases in Rcr1-mediated resistance response.This study,the first of its kind,provides valuable insights into the functionality of Rcr1.Further,the new vector p HHIGR-Hsp18.2 demonstrated an inducible feature on the removal of add-on traits,which should be useful for functional genomics and other similar research in brassica crops.

Use of gene-editing technology to introduce targeted modifications in pigs

Pigs are an important resource in agriculture and serve as a model for human diseases. Due to their physiological and anatomical similarities with humans, pigs can recapitulate symptoms of human diseases, making them a useful model in biomedicine. However, in the past pig models have not been widely used partially because of the difficulty in genetic modification. The lack of true embryonic stem cells in pigs forced researchers to utilize genetic modification in somatic cells and somatic cell nuclear transfer(SCNT) to generate genetically engineered(GE) pigs carrying site-specific modifications. Although possible, this approach is extremely inefficient and GE pigs born through this method often presented developmental defects associated with the cloning process. Advancement in the gene-editing systems such as Zinc-Finger Nucleases(ZFNs), Transcription activator-like effector nucleases(TALENs), and the Clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated 9(Cas9) system have dramatically increased the efficiency of producing GE pigs. These gene-editing systems, specifically engineered endonucleases, are based on inducing double-stranded breaks(DSBs) at a specific location, and then site-specific modifications can be introduced through one of the two DNA repair pathways: non-homologous end joining(NHEJ) or homology direct repair(HDR).Random insertions or deletions(indels) can be introduced through NHEJ and specific nucleotide sequences can be introduced through HDR, if donor DNA is provided. Use of these engineered endonucleases provides a higher success in genetic modifications, multiallelic modification of the genome, and an opportunity to introduce site-specific modifications during embryogenesis, thus bypassing the need of SCNT in GE pig production. This review will provide a historical prospective of GE pig production and examples of how the gene-editing system, led by engineered endonucleases, have improved GE pig production. We wil also present some of our current progress related to the optimal use of CRISPR/Cas9 system during embryogenesis.

Designing and generating a mouse model:frequently asked questions

Genetically engineered mouse(GEM)models are commonly used in biomedical research.Generating GEMs involve complex set of experimental procedures requiring sophisticated equipment and highly skilled technical staff.Because of these reasons,most research institutes set up centralized core facilities where custom GEMs are created for research groups.Researchers,on the other hand,when they begin thinking about generating GEMs for their research,several questions arise in their minds.For example,what type of model(s)would be best useful for my research,how do I design them,what are the latest technologies and tools available for developing my model(s),and finally how to breed GEMs in my research.As there are several considerations and options in mouse designs,and as it is an expensive and time-consuming endeavor,careful planning upfront can ensure the highest chance of success.In this article,we provide brief answers to several frequently asked questions that arise when researchers begin thinking about generating mouse model(s)for their work.

Mutation of cytotoxin-associated gene A affects expressions of antioxidant proteins of Helicobacter pylori

AIM: To determine if disruption of the cagA gene of Helicobacter pylori (H pylori) has an effect on the expression of other proteins at proteome level. METHODS: Construction of a cagA knock out mutant Hp27 _△cagA (cagA -) via homologous recombination with the wild-type strain Hp27 (cagA+) as a recipient was performed. The method of sonication-urea-CHAPS-DTT was employed to extract bacterial proteins from both strains. Soluble proteins were analyzed by two-dimensional electrophoresis (2-DE). Images of 2-DE gels were digitalized and analyzed. Only spots that had a statistical signif icance in differential expression were selected and analyzed by matrix-assisted laser desorption/ionizationtime of flight mass spectrometry (MALDI-TOF-MS). Biological information was used to search protein database and identify the biological function of proteins. RESULTS: The proteome expressions between wild-type strain and isogenic mutant with the cagA gene knocked-out were compared. Five protein spots with high abundance in bacteria proteins of wild-type strains, down-regulated or absently expressed in bacteria proteins of mutants, were identified and analyzed. From a quantitative point of view, the identified proteins are related to the cagA gene and important antioxidant proteins of H pylori , including alkyl hydroperoxide reductase (Ahp), superoxide dismutase (SOD) and modulator of drug activity (Mda66), respectively, suggesting that cagA is important to maintain the normal activity of antioxidative stress and ensure H pylori persistent colonization in the host. CONCLUSION: cagA gene is relevant to the expressions of antioxidant proteins of H pylori, which may be a novel mechanism involved in H pylori cagA pathogenesis.

Improve Ethanol Yield Through Minimizing Glycerol Yield in Ethanol Fermentation of Saccharomyces cerevisiae

In ethanol fermentation of Saccharomyces cerevisiae （S. cerevisiae）, glycerol is one of the main by-products. The purpose of this investigation was to increase ethanol yield through minimizing glycerol yield by using mutants in which FPS1 encoding a channel protein that mediates glycerol export and GPD2 encoding one of glycerol-3-phosphate dehydrogenase were knocked-out using one-step gene replacement. GLT1 and GLN1 that encode glutamate synthase and glutamine synth.etase, respectively,were overexpressed using two-step gene replacment in fpsl△gpd2△ mutant.The fermentation properties of ZAL69（fpsl△：：LEU2 gpd2△：：URA3） and ZAL808 （fps1△：：LEU2 gpd2△：：URA3 PPGK1-GLT1 PPGK1-GLN1） under microaerobic conditions were investigated and compared with those of wild type（DC124）. Consumption of glucose, yield of ethanol, yield of glycerol, acetic acid, and pyruvic acid were monitored. Compared with wild type, the ethanol yield of ZAL69 and ZAL808 were improved by. 13.17% and 6.66 %, respectively, whereas glycerol yield decreased by 37.4 % and 41.7 %. Meanwhile, acetic acia yield and pyruvic acid yield aecreasea aramatlcally comparea to wild type. Our results indicate that FPS1 and GPD2 deletion of S. cerevisiae resulted in reduced glycerol yield and increased ethanol yield, but simultaneous overexpression of GLT1 and GLN1 infps1△gpd2△ mutant did not have a higher ethanol yield thanfps1△gpd2△ mutant.

The collagen type Ⅰ alpha 1 chain gene is an alternative safe harbor locus in the porcine genome

Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors.Safe harbor loci in the animal genome enable consistent overexpression of foreign genes,without side effects.However,relatively few safe harbor loci are available in pigs,a fact which has impeded the development of multi-transgenic pig research.We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain(COL1A1)gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)system.After the knock-in of a 2A peptide-green fluorescence protein(2A-GFP)transgene in the last codon of COL1A1 in multiple porcine cells,including porcine kidney epithelial(PK15),porcine embryonic fibroblast(PEF)and porcine intestinal epithelial(IPI-2I)cells,quantitative PCR(qPCR),Western blotting,RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus.The qPCR results showed that the GFP knock-in had no effect(P=0.29,P=0.66 and P=0.20 for PK15,PEF and IPI-2I cells,respectively)on the mRNA expression of COL1A1 gene.Similarly,no significant differences(P=0.64,P=0.48 and P=0.80 for PK15,PEF and IPI-2I cells,respectively)were found between the GFP knock-in and wild type cells by Western blotting.RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation(P<2.2e–16)with that of the wild type cells,indicating that the GFP knock-in did not alter the global expression of endogenous genes.Furthermore,the CCK8 assay showed that the GFP knock-in events had no adverse effects(P_(24)h=0.31,P_(48)h=0.96,P_(72)h=0.24,P_(96)h=0.17,and P_(120)h=0.38)on cell proliferation of PK15 cells.These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment.

CB1 receptor activation on VgluT2-expressing glutamatergic neurons underlies Δ9-tetrahydrocannabinol(Δ9-THC)-induced aversive effects in mice

OBJECTIVE Cannabis can be rewarding or aversive.Cannabis reward is believed to be mediated by activation of cannabinoid CB1 receptors(CB1 Rs) on GABAergic neurons that disinhibit dopaminergic neurons in the ventral tegmental area(VTA).However,little is known about the mechanisms underlying cannabis aversion in rodents.Our study aimed at dig the mechanisms underlying cannabis aversion.METHODS We first created CB1-floxed mice and then generated conditional CB1-knockout mice(VgluT2-CB1-/-) in glutamatergic neurons that express vesicular glutamate transporter 2(VgluT2).We then used immunohistochemistry and RNAscope in situ hybridization assays to examine whether CB1 Rs are expressed in VTA GABAergic neurons and glutamatergic neurons.We also used Cre-dependent viral vector to express light-sensitive channelrhodopsin-2 into VTA glutamatergic neurons.Next,conditioned place preference and intracranial self-stimulation(ICSS) maintained by optical activation of VTA glutamatergic neurons were employed to evaluate the effects of Δ9-THC on brain reward function.RESULTS CB1 Rs are found not only on VTA GABAergic neurons,but also on VTA glutamatergic neurons that express vesicular glutamate transporter 2(VgluT2).Photoactivation of VTA glutamatergic neurons produced robust intracranial self-stimulation(ICSS) behavior,which was dose-dependently blocked by DA receptor antagonists,but enhanced by cocaine.In contrast,Δ9-tetrahydrocannabinol(Δ9-THC),the major psychoactive component of cannabis,produced dose-dependent conditioned place aversion and a reduction in the above optical ICSS in VgluT2-cre control mice,but not in VgluT2-CB1-/-mice.CONCLUSION Activation of CB1 Rs in VgluT2-expressing glutamate neurons produces aversive effects that might explain why cannabinoid is not rewarding in rodents and might also account for individual differences in the hedonic effects of cannabis in humans.

The Transactivating Function 2 (AF-2) of Estrogen Receptor (ER) <i>α</i>Is Indispensable for ER<i>α</i>-Mediated Physiological Responses and AF-1 Activity

Estrogen has various physiological functions and the estrogen receptor (ER) is a key regulator of those functions. ERα is a ligand-dependent transcription factor and that activity is mediated by the transactivating function-1 (AF-1) in the N-terminal domain and transactivating function-2 (AF-2) in the C-terminal ligand-binding domain. The functions of ERα AF-1 and AF-2 have been characterized by various in vitro experiments, however, there is still less information about the in vivo physiological functions of ERα AF-1 and AF-2. Recently, we established a genetically mutated ERα AF-2 knock-in mouse (AF2ERKI) that possessed L543A, L544A mutated-ERα. This AF-2 core mutation disrupted AF-2 function and resulted in ERα null phenotypes. This mouse model revealed that proper AF-2 core structure and function were indispensable for ERα-mediated physiological responses and AF-1 functionality. AF2ER mutation reverses the ERα antagonists to agonists and that activity is mediated by AF-1 solely. The pure antagonist, ICI182780/fulvestrant, activated several estrogen-mediated physiological responses in the AF2ERKI mouse. The AF2ERKI mouse model will be useful to discern estrogen physiological functions which involve AF-1.

TALEN-Mediated FLAG-Tagging of Endogenous Histone Methyltransferase DOT1L

Histone modification including H3 lysine 79 methylation (H3K79me) plays a key role during gene transcription and DNA damage repair. DOT1L, the sole methyltransferase for three states of H3K79me, is implicated in leukemia, colorectal cancer, and dilated cardiomyopathy. However, understanding of DOT1L and H3K79me in these pathways and disease pathogenesis has been limited due to the difficulty of working with DOT1L protein. For instance, locus-specific or genome-wide binding sites of DOT1L revealed by chromatin immunoprecipitation (ChIP)-based methods are necessary for inferring its functions, but high-quality ChIP-grade antibodies are currently not available. Herein we have developed a knock-in approach to tag endogenous DOT1L with 3 × Flag at its C-terminal domain to follow functional analyses. The knock-in was facilitated by using TALENs to induce a targeted double-strand break at the endogenous DOTIL to stimulate local homologous recombination at that site. The single cell colonies with successful knock-in were isolated and verified by different methods. We also demonstrated that tagged DOT1L maintains its normal function in terms of methylation and that the engineered cells would be very useful for further studies.

Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process.Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline.Previous pig models with tetracycline regulatory elements were generated through random integration.This process often resulted in uncertain expression and unpredictable phenotypes,thus hindering their applications.Here,by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus,respectively,a double knock-in reporter pig model was generated.We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo.Two att P sites were arranged to flank the td Tomato to switch reporter gene.Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos.To display the flexible application of this system,we generated a pig strain with Dox-inducing h KRASexpression through phiC31 integrase-mediated cassette exchange.After eight months of Dox administration,squamous cell carcinoma developed in the nose,mouth,and scrotum,which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis.Overall,the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.

Identification in Chinese patients with GLIALCAM mutations of megalencephalic leukoencephalopathy with subcortical cysts and brain pathological study on Glialcam knock-in mouse models

Background Megalencephalic leukoencephalopathy with subcortical cysts(MLC)is a rare neurological degenerative disorder caused by the mutations of MLC1 or GLIALCAM with autosomal recessive or autosomal dominant inheritance and a different prognosis,characterized by macrocephaly,delayed motor and cognitive development,and bilateral abnormal signals in cerebral white matter(WM)with or without cysts on magnetic resonance imaging(MRI).This study aimed to reveal the clinical and genetic features of MLC patients with GLIALCAM mutations and to explore the brain pathological characteristics and prognosis of mouse models with different modes of inheritance.Methods Clinical information and peripheral venous blood were collected from six families.Genetic analysis was performed by Sanger sequencing of GLIALCAM.Glialcam^(Arg92Trp/+)and Glialcam^(Lys68Met/Thr132Asn)mouse models were generated based on mutations from patients(c.274C>T(p.Arg92Trp)(c.203A>T(p.Lys68Met),and c.395C>A(p.Thr132Asn))).Brain pathologies of the mouse models at different time points were analyzed.Results Six patients were clinically diagnosed with MLC.Of the six patients,five(Pt1-Pt5)presented with a heterozygous mutation in GLIALCAM(c.274C>T(p.Arg92Trp)or c.275G>C(p.Arg92Pro))and were diagnosed with MLC2B;the remaining patient(Pt6)with two compound heterozygous mutations in GLIALCAM(c.203A>T(p.Lys68Met)and c.395C>A(p.Thr132Asn))was diagnosed with MLC2A.The mutation c.275C>G(p.Arg92Pro)has not been reported before.Clinical manifestations of the patient with MLC2A(Pt6)progressed with regression,whereas the course of the five MLC2B patients remained stable or improved.The Glialcam^(Arg92Trp/+)and Glialcam^(Lys68Met/Thr132Asn)mouse models showed vacuolization in the anterior commissural WM at 1 month of age and vacuolization in the cerebellar WM at 3 and 6 months,respectively.At 9 months,the vacuolization of the GlialcamiLys68Met/Thr132Asn mouse model was heavier than that of the Glialcam^(Arg92Trp/+)mouse model.Decreased expression of Glialcam in Glialcam^(Arg92Trp/+)and Glialcam^(Lys68Met/Thr132Asn)mice may contribute to the vacuolization.Conclusions Clinical and genetic characterization of patients with MLC and GLIALCAM mutations revealed a novel mutation,expanding the spectrum of GLIALCAM mutations.The first Glialcam mouse model with autosomal recessive inheritance and a new Glialcam mouse model with autosomal dominant inheritance were generated.The two mouse models with different modes of inheritance showed different degrees of brain pathological features,which were consistent with the patients'phenotype and further confirmed the pathogenicity of the corresponding mutations.

LRRK2 mutant knock-in mouse models: therapeutic relevance in Parkinson’s disease

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are one of the most frequent genetic causes of both familial and sporadic Parkinson’s disease (PD). Mounting evidence has demonstrated pathological similarities between LRRK2-associated PD (LRRK2-PD) and sporadic PD, suggesting that LRRK2 is a potential disease modulator and a thera-peutic target in PD. LRRK2 mutant knock-in (KI) mouse models display subtle alterations in pathological aspects that mirror early-stage PD, including increased susceptibility of nigrostriatal neurotransmission, development of motor and non-motor symptoms, mitochondrial and autophagy-lysosomal defects and synucleinopathies. This review provides a rationale for the use of LRRK2 KI mice to investigate the LRRK2-mediated pathogenesis of PD and implications from current findings from different LRRK2 KI mouse models, and ultimately discusses the therapeutic potentials against LRRK2-associated pathologies in PD.

Using Huntingtin Knock-In Minipigs to Fill the Gap Between Mouse Models and Patients with Huntington's Disease

Huntington＇s disease （HD） is an inherited autosomal dominant neurodegenerative disease characterized by pro- gressive motor deficits, cognitive decline, and psychiatric symptoms. It is caused by a pathological expansion of CAG trinucleotide repeats in exon 1 of the HD gene, resulting in the translation of a mutant form of huntingtin protein （mutant Htt） with an expanded polyglutamine domain in the N-terminal region [1 ]. Despite great progress in understanding the pathogenesis of HD using multiple mouse models, the exact mechanisms by which mutant Htt induces neuronal dysfunction and death are still not completely clear, and there is no curative treatment for this disease. An important reason is that the mouse, which is the most widely used animal model in HD research, differs from the human in many aspects, including the physiology, drug metabolism, blood-brain barrier, life span, brain volume, and neuroanatomical organization [2]. Thus, it is necessary to establish HD models with higher species than rodents, such as the dog, pig, and non- human primate, so as to bridge the gap between preclinical mouse models and clinical studies.

Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System

Transcription activator-like effector nucleases （TALENs） and clustered regularly interspaced short palindromic repeats （CRISPR）/ CRISPR-associated （Cas） systems have emerged as powerful tools for genome editing in a variety of species. Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmlPKIA, ZmlPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmlPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition, the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies （13.1%） similar to those obtained with TALENs （9.1%）. Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize.

TMEM43-S358L mutation enhances NF-κBTGFp signal cascade in arrhythmogenic right ventricular dysplasia/cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy(ARVD/C)is a genetic cardiac muscle disease that accounts for approximately 30%sudden cardiac death in young adults.The Ser358Leu mutation of transmembrane protein 43(TMEM43)was commonly identified in the patients of highly lethal and fully penetrant ARVD subtype,ARVD5.Here,we generated TMEM43 S358L mouse to explore the underlying mechanism.This mouse strain showed the classic patholo.gies of ARVD patients,including structural abnormalities and cardiac fibrofatty.TMEM43 S358L mutation led to hyper-activated nuclear factor kB(NFkB)activation in heart tissues and primary cardiomy.ocyte cells.Importantly,this hyper activation of NF-κB directly drove the expression of pro-fibrotic gene,transforming growth factor beta(TGFβ),and enhanced downstream signal,indicating that TMEM43 S358L mutation up-regulates NF-κB-TGFβ signal cascade during ARVD cardiac fibrosis.Our study partially reveals the regulatory mechanism of ARVD development.

p53-dependent and independent apoptosis induced by ionizing radiation in murine bone marrow cells

APOPTOSIS or programmed cell death is a model of cell death, along with typical morphological and biochemical changes. The cell death, occurring during embryonic development, T and B cell maturation and endocrine-induced atrophy, is a physiological apoptosis which controls the amount of cells in the body. Apoptosis can also be induced by various factors, such as ionizing radiation and anti-tumour agents which damage DNA, and has been proposed to be a consequence of the induced expression of some tumour-related genes or increase of the proteins sta-