Turning Control of a Multilink Biomimetic Robotic Fish

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转基因英语作文120字Title: The Pros and Cons of Genetic Modification。

Genetic modification, often referred to as genetic engineering or GMOs (Genetically Modified Organisms), has sparked intense debates globally. Proponents argue that it offers numerous benefits, while critics raise concerns about potential risks. 。

On the one hand, genetic modification holds promise for enhancing crop yield and quality. By introducing traits such as pest resistance and drought tolerance, scientists aim to create more resilient crops capable of withstanding environmental challenges. This could lead to increased food security, particularly in regions prone to agricultural setbacks.Moreover, genetic modification has the potential to address nutritional deficiencies. Through biofortification, essential nutrients can be incorporated into staple crops,improving public health outcomes, especially in developing countries where malnutrition remains a significant issue.Furthermore, genetic modification can contribute to sustainable agriculture practices. By reducing the need for chemical pesticides and fertilizers, it can mitigate environmental damage and promote eco-friendly farming methods.On the other hand, critics raise valid concerns regarding the long-term impacts of genetic modification. One major worry is the potential for unintended consequences on ecosystems and human health. The introduction of modified genes into the environment could disrupt natural ecosystems and lead to the emergence of superweeds or pests resistant to current control methods.Additionally, there are ethical considerations surrounding genetic modification, particularly in the realm of human genetic engineering. The ability to manipulate the genetic makeup of organisms raises questions about the boundaries of scientific intervention and the potential formisuse or unintended consequences.In conclusion, genetic modification presents both opportunities and challenges. While it offers the potential for improving crop resilience, nutritional value, and sustainability, it also raises concerns about environmental and health impacts, as well as ethical dilemmas. Moving forward, a balanced approach that considers both the benefits and risks is essential to harnessing the full potential of genetic modification while minimizing its drawbacks.。

Endonucleases new tools to edit the mouse genome

ReviewEndonucleases:new tools to edit the mouse genome ☆Tobias Wijshake a ,Darren J.Baker b ,Bart van de Sluis a ,⁎a Molecular Genetics,University of Groningen,University Medical Center Groningen,Antonius Deusinglaan 1,9713AV Groningen,The Netherlands bDepartment of Pediatric and Adolescent Medicine,Mayo Clinic College of Medicine,200First St SW,Rochester,MN 55905,USAa b s t r a c ta r t i c l e i n f o Article history:Received 20January 2014Received in revised form 16April 2014Accepted 18April 2014Available online 30April 2014Keywords:Genome editing MouseEndonucleases ZFN TALENCRISPR/CasMouse transgenesis has been instrumental in determining the function of genes in the pathophysiology of human diseases and modi ﬁcation of genes by homologous recombination in mouse embryonic stem cells remains a widely used technology.However,this approach harbors a number of disadvantages,as it is time-consuming and quite laborious.Over the last decade a number of new genome editing technologies have been developed,including zinc ﬁnger nucleases (ZFNs),transcription activator-like effector nucleases (TALENs)and clustered reg-ularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas).These systems are characterized by a designed DNA binding protein or RNA sequence fused or co-expressed with a non-speci ﬁc endonuclease,respectively.The engineered DNA binding protein or RNA sequence guides the nuclease to a speci ﬁc target se-quence in the genome to induce a double strand break.The subsequent activation of the DNA repair machinery then enables the introduction of gene modi ﬁcations at the target site,such as gene disruption,correction or in-sertion.Nuclease-mediated genome editing has numerous advantages over conventional gene targeting,includ-ing increased ef ﬁciency in gene editing,reduced generation time of mutant mice,and the ability to mutagenize multiple genes simultaneously.Although nuclease-driven modi ﬁcations in the genome are a powerful tool to generate mutant mice,there are concerns about off-target cleavage,especially when using the CRISPR/Cas sys-tem.Here,we describe the basic principles of these new strategies in mouse genome manipulation,their inherent advantages,and their potential disadvantages compared to current technologies used to study gene function in mouse models.This article is part of a Special Issue entitled:From Genome to Function.©2014Elsevier B.V.All rights reserved.1.IntroductionGenome-wide association studies (GWAS)have been instrumental in the identi ﬁcation of single nucleotide polymorphisms (SNP)s associ-ated with complex human diseases.The number of genetic associations has been steadily increasing each year since the introduction of this approach in 2005.Genomic regions marked by speci ﬁc SNPs have attracted the attention of many researchers to potentially identifying the causal variant and understanding the pathophysiology of the disease [1,2].These genomic regions can contain either protein-coding (direct protein variants)or non-coding regions that might regulatethe expression of genes.However,discovering the causal variant and re-vealing the underlying biological mechanism of the associated disease is still a complicated process.For a number of reasons,the mouse is the most valuable and readily accessible animal model as a biological source to study genes within the candidate loci.The genome of the mouse has been fully sequenced,and most of the genes (~99%)in human are also present in mice.Mice are highly comparable to humans with respect to organs,tissues and physiological systems,enabling the study of gene-environment interactions in the whole organism.Furthermore,mice are easy to breed with a relatively short generation time,are small,and can be housed together,thereby keeping the costs relatively low.The discovery of gene editing via homologous recombination in mouse embryonic stem (ES)cells has further spurred the use of mice over other animal models [3–5].Here,we will give an overview of the various tools for gene modi ﬁcation that have been developed during the last decades.Additionally,we will focus on new developments in mouse technology and the advantages these have over existing technol-ogies to translate genetic ﬁndings into functional biological assessments.2.Gene editing by homologous recombinationMost human diseases are studied from a candidate gene approach that has been identi ﬁed by linkage or association studies,or deepBiochimica et Biophysica Acta 1842(2014)1942–1950Abbreviations:CRISPR/Cas,clustered regularly interspaced short palindromic repeats/CRISPR-associated;crRNA,CRISPR RNA;DSB,double-strand break;dsDNA,double-strand DNA;ES,embryonic stem;FLASH,fast ligation-based automatable solid-phase high-throughput;gRNA,guide RNA;GWAS,genome-wide association studies;HDR,homology-directed repair;iPS,induced pluripotent stem;NHEJ,non-homologous end joining;OPEN,oligomerized pool engineering;PAM,protospacer adjacent motif;RVD,re-peat variable di-residue;SELEX,systematic evolution of ligands by exponential enrich-ment;SNP,single nucleotide polymorphism;SpCas9,S.pyogenes Cas9;TALEN,transcription activator-like effector nuclease;tracrRNA,trans-activating crRNA;ZFN,zinc ﬁnger nuclease;ZFP,zinc ﬁnger protein☆This article is part of a Special Issue entitled:From Genome to Function.⁎Corresponding author.Tel.:+31503638158;fax:+31503638971,+31503619911.E-mail address:A.J.A.van.de.Sluis@umcg.nl (B.van deSluis)./10.1016/j.bbadis.2014.04.0200925-4439/©2014Elsevier B.V.All rightsreserved.Contents lists available at ScienceDirectBiochimica et Biophysica Actaj ou r n a l h o m e p a g e :w ww.e l s e v i e r.c om /l o c a t e /bb a d i ssequencing approaches[6,7].Although human diseases are usually very complex,typically involving gene-gene and/or gene-environment in-teractions,the most straightforward and commonly used method to study the function of candidate genes is by modifying these genes in mice.The development of gene targeting technology in ES cells was a major breakthrough that led to the generation of numerous mutant mouse models.The technique makes use of homologous recombination to mutagenize the genome in ES cells,which creates a deletion, insertion,or point mutation[8].However,~30%of all knockouts are embryonic or early postnatal lethal,which led to the development of other mutagenesis strategies,like Dre/Rox,Flp/Frt and the most widely used Cre/LoxP system.These systems provide the possibility of generating a tissue/cell-speciﬁc gene knockout(discussed below) [9–11].Cre/LoxP is a site-speciﬁc recombination system that was dis-covered in bacteriophage P1[11,12].Cre recombinase drives recombi-nation between two DNA recognition sites of34bp,also known as LoxP sites[8].Genomic regions that areﬂanked by loxP sites in the same orientation,also termed a“ﬂoxed allele”,will be excised in cells expressing Cre recombinase[13].In general,this mutagenesis approach is commonly used if the gene of interest is vital for normal embryogenesis or if there is a necessity to investigate the function of the gene in a tissue/cell-speciﬁc context. Mice carrying theﬂoxed alleles will be crossed with a mouse strain con-taining a transgene encoding the Cre recombinase under the control of a tissue-speciﬁc promoter,which results in conditional/tissue-speciﬁc knockout mice[14–16].Over the last two decades,numerous,tissue-speciﬁc,Cre-driver lines have been developed.However,some drawbacks in the Cre/LoxP system have also started to emerge,which was recently extensively reviewed[16,17].One major concern is the tissue-speciﬁcity of the chosen promoter that drives the Cre transgene. Expression of various genes assumed to be restricted to a speciﬁc tissue or cell type are actually expressed in multiple tissues/cells[16–18].An additional problem is that Cre recombinase transgenic mice can have too high or low Cre activity,leading to toxicity or inefﬁcient deletion of the gene,respectively[16].Furthermore,Cre recombinase itself can also cause unwanted side-effects,such as random recombination,re-duced proliferation and increased apoptosis,supporting the need to in-clude the Cre recombinase transgenic mice as an additional control in the study design[16].Another elegant method to examine gene function in a more physi-ological fashion is by engineering mice with reduced expression of the gene of interest.This can be accomplished by creating a hypomorphic allele that results in the expression of only a fraction of the normal pro-tein bining a hypomorphic allele with either a wild-type, knockout,or hypomorphic allele enables generation of a series of mice with a gradual reduction in protein levels[19].For example,this strate-gy has successfully been used to study the mitotic checkpoint proteins BubR1and plete ablation of these genes results in embryonic lethality,but mice with reduced protein levels are born healthy and show an overt phenotype later in life[20,21].The strategies to generate a hypomorphic allele have recently been described in detail[19,22].Although gene editing by homologous recombination in ES cells is still the most widely used strategy to generate mutant mice the efﬁcien-cy of homologous recombination is very low.Therefore this genetic editing method has to be performed in ES cellsﬁrst instead of in the mouse directly.In addition,the availability of ES cells from different spe-cies is limited.All this combined has led to the development of new techniques,such as ZFNs,TALENs and CRISPR/Cas that harbor signiﬁ-cantly improved efﬁciencies in gene editing[23–25].The basic princi-ples and advantages of these technologies will be discussed in the following sections.3.Zincﬁnger nucleasesZincﬁnger nucleases(ZFNs)facilitate genetic modiﬁcation through the introduction of a double strand break(DSB)in a DNA sequence of interest.Subsequent DNA break repair then enables the introduction of the desired modiﬁcation,which is discussed in detail below[26,27]. The DSB is produced by a ZFN,which is a sequence-speciﬁc endonucle-ase that can be designed to cleave at a precise DNA sequence[27].A ZFN consists of a varying number of zincﬁnger proteins(ZFPs)or Cys2His2ﬁngers which are usually fused to the nuclease domain of Fok I,a restric-tion enzyme that cleaves non-speciﬁc DNA sequences[27–31](Fig.1A). Each ZFP is able to recognize a distinct three-base-pair DNA sequence and a typical ZFN consists of3–6fused zincﬁnger proteins.Optimal Fok I cleavage by ZFNs requires two independent ZFNs to bind on oppo-site DNA strands in the appropriate orientation and at the correct distance from each other[27,32](Fig.1A).The introduction of a DSB by ZFNs at a predeﬁned DNA locus pro-vokes activation of a conserved DNA repair pathway,namely non-homologous end joining(NHEJ)or homology-directed repair(HDR) [33–35](Fig.1B).In most cases the DSB is repaired by the NHEJ pathway, which efﬁciently ligates the two broken ends.However,the NHEJ path-way is error-prone and the repair can result in small deletions and/or in-sertions(indels),which can lead to gene disruption[27,33](Fig.1B). Gene inactivation was initially applied by expression of two ZFNs direct-ed against the yellow gene in the larvae of Drosophila melanogaster, which resulted in germline mutations[36,37].Subsequently,ZFN tech-nology has successfully been applied to mutagenize genes in various or-ganisms,including zebraﬁsh,rats and mice with varying frequency[23, 38–42].For example,microinjection of engineered ZFNs in embryos was used to generate Mrd1a and Tnfrsf9knockout mice,respectively[23,42]. In addition to single gene disruption,ZFN technology has also been used to target two or three genes simultaneously in mammalian cells[43,44]. Furthermore,larger deletions,translocations,duplications and inver-sions can be introduced with ZFN[44–48].HDR enables the introduction of single nucleotide changes(gene correction)after DSB induction by ZNF upon simultaneous delivery of a donor DNA repair template,which contains homology armsﬂanking the site of alteration[37,49,50](Fig.1B).This opens the possibility to study the functional consequences of human disease-associated point mutations in the preferred cells and/or model organisms[23,51–54]. In addition,this approach can be used to engineer larger modiﬁcations, including insertions of loxP sites,ﬂuorescent proteins,antibiotic resis-tance markers,or other tags[52,55–59].There are limitations to gene correction and gene addition via HDR:the need for co-delivery of a de-signed DNA donor template together with a speciﬁc-ZFN,and the strong preference of a cell for NHEJ over HDR-mediated repair of the DSB.Pos-sible solutions are either to use ZFN nickases or a vector carrying multi-ple copies of linear donor fragments,which both increase HDR-driven genome editing while reducing unwanted mutations caused by NHEJ [60–63].Importantly,ZFN-mediated gene modiﬁcation has great therapeutic potential.ZFN has the advantage over known knockdown or blocking strategies because it is efﬁcient and persistent,which could avoid the need for life-long treatment.For example,independent studies have shown that disruption of the CCR5and CXCR4gene,which encode HIV co-receptors,protects against HIV-1infection in vitro and in vivo. Based on the CCR5studies,ZFN-mediated therapies for HIV have been designed and are currently being used in Phase2clinical trials[27, 64–68].ZFN-induced HDR can also be exploited to correct genetic disease-causing mutations,as demonstrated in human induced pluripo-tent stem(iPS)cells carrying mutations underlying Parkinson’s disease,α1-antitrypsin deﬁciency,or sickle-cell anemia[69–71].Furthermore, ZFN-driven gene correction has been demonstrated to be effective in a mouse model of hemophilia,raising the possibility of in vivo genome editing by ZFN as a strategy for the treatment of genetic diseases[72]. The risk for potential off-target DNA cleavage when using ZFN technol-ogy raises some concerns.Increased ZFN speciﬁcity and simultaneous reduction of off-target cleavage can be achieved by linking more ZFPs in a ZFN,optimizing the orientation of protein-DNA interaction and using a heterodimer ZFN pair[51,73].Although some reports have1943T.Wijshake et al./Biochimica et Biophysica Acta1842(2014)1942–1950demonstrated increased cytotoxicity issues when using heterodimers [50,74].A systematic evolution of ligands by exponential enrichment (SELEX)protocol and unbiased genome-wide analysis can be utilized to determine the speciﬁcity for ZFP DNA binding and rank the risk of potential off-target sites[75,76].Combination of the SELEX protocol with ultradeep sequencing conﬁrmed the high ZFN speciﬁcity for the target site in CCR5and only identiﬁed rare off-target sites with low frequency[64].There are currently a number of methods to fuse ZFPs with each other to generate new ZFNs,including modular assembly[77], Oligomerized Pool ENgineering(OPEN)system[78],a bacterial one-hybrid system[39],twoﬁnger modules[79,80]and context-dependent assembly[81].However,the construction of ZFNs is challenging for non-specialist laboratories,especially with the bacterial one-hybrid and OPEN systems.Furthermore,most ZFNs fail to modify the gene of interest in vivo.This can be caused by either speciﬁcity issues through identical or very similar sequences in the ge-nome or by the chromatin structure at the site of interest that prevents ZFN binding[27].Current studies are focusing on improving the design and construction of ZFNs,increasing their speciﬁcity and thereby reducing off-target cleavage.In conclusion,ZFN technology appears to be a promising mutagenesis tool for generating mutant animals,includ-ing mice,and may have the potential to be used in therapeutic applications.4.Transcription activator-like effector nucleases(TALENs)Transcription activator-like effectors(TALEs)from plant pathogenic Xanthomas are virulence factors secreted via the type III system that can bind to host DNA and activate expression of effector-speciﬁc host genes [82,83].TALEs contain a characteristic central domain of DNA-binding tandem repeats,a nuclear localization signal,and a C-terminal tran-scriptional activation domain[84–86].A typical repeat is33–35amino acids in length and contains two hypervariable amino acid residues at positions12and13,known as the“repeat variable di-residue”(RVD) (Fig.2A).Two studies discovered that an RVD is able to recognize one speciﬁc DNA base pair and that sequential repeats match consecutive DNA sequences.They demonstrated that target DNA speciﬁcity is based on the simple code of the RVDs,which thus enables prediction of target DNA sequences[82,83].Nucleotide speciﬁcity of repeats was shown for RVDs encoding NN,NI,HD and NG for recognition of guanine, adenine,cytosine and thymine,respectively[83,87].Theﬁrst successful generation of TALE nucleases(TALENs)was reported in two studies,in which they fused either native or modiﬁed TALEs to the catalytic do-main of the Fok I restriction enzyme.These native and custom-made TALEN fusions were able to induce target-speciﬁc double-strand cleav-age in yeast[88,89].The essential TALEN architecture necessary for efﬁ-cient genome editing in human cells was determined by linking TALE truncation variants to the catalytic domain of Fok I.This approachA T GCT T T T TTTTC C G A A GG A CCG G GGGCCCCCAAAAAGAG G G GGT T T TTTC CC CA A A AAC C GTAFok IFok IZF ZF ZFZFZFZF5’3’3’5’ABZFN cleavageNon-HomologousEnd JoiningGene disruptionAdd DNA donorHomology-DirectedRepairGene correctionGene additionFig.1.Overview of ZFNs.A.ZFN consisting of three zincﬁnger proteins fused to the catalytic domain of Fok I restriction enzyme.Each zincﬁnger protein is able to bind to three nucleotides and can guide the ZFN to a speciﬁc target site in the genome.Two ZFNs targeting a speciﬁc sequence on opposite sides of genomic DNA are necessary to allow dimerization of two non-speciﬁc Fok I nucleases.B.Dimerization of two Fok I enzymes induces a DSB at the target site.Subsequent DNA-repair by the erroneous NHEJ pathway can introduce the desired genomic modiﬁcation,like gene disruption.Alternatively,the addition of a DNA repair template can facilitate HDR-mediated genome editing and result in gene correction or addition.ZF=zinc ﬁnger protein.1944T.Wijshake et al./Biochimica et Biophysica Acta1842(2014)1942–1950revealed that the N-terminal 152residues were dispensable and TALE variants containing 28or 63of the 278original C-terminal amino acids were suf ﬁcient to drive ef ﬁcient gene modi ﬁcations of two endogenous genes.Furthermore,they demonstrated that the correct distance between the TALEN pair is essential for successful cleavage [87].Similarly to ZFNs,TALENs enable genetic modi ﬁcation through induction of a double strand break (DSB)in a DNA target sequence.Ensuing DNA break repair by either the NHEJ or the HDR-mediated pathway can be exploited to introduce the desired modi ﬁcation (e.g.gene disruption,gene correction or gene insertion)[24](Fig.2B).TALENs have been utilized to ef ﬁciently introduce targeted genetic modi ﬁcations in a number of model organisms,including Drosophila melanogaster [90],zebra ﬁsh [91–93],rat [94],pig and cow [95],rhesus and cynomolgus monkeys [96].Most of these studies used TALENs to generate an NHEJ-mediated knockout animal,but two studies reported the use of two TALEN pairs to generate larger deletions and inversions in livestock ﬁbroblasts and zebra ﬁsh [95,97].Two designed TALEN pairs were also able to induce cancer-relevant translocations found in ana-plastic large cell lymphoma [98].Injection of TALEN mRNA speci ﬁc for exon 2of Pibf1gene and for exon 1of Sepw1gene into the cytoplasm of mouse pronuclear-stage embryos resulted in founders carrying null mutations in the Pibf1and Sepw1gene.All mutations observed in F 0mice were transmitted through the germline [99].Increasing the amount of mRNA injected can produce a higher mutation rate and bi-allelic mutation frequency,but this can also result in fewer mutant mice due to the toxicity of high doses of mRNA [99].Similar approaches using microinjection of TALEN mRNA targeting genes in mouse oocytes and zygotes have been successful in engineering knockout mice [100–105].Exploitation of TALEN-mediated genome editing in mouse ES cells resulted in mice with targeted gene disruptions and insertions in two Y chromosome-linked genes,which was previously impossible with conventional gene-targeting technology [106].HDR-driven inser-tion was introduced by simultaneous delivery of TALEN with a designed single-stranded DNA repair template in human pluripotent stem cells,somatic cells,zebra ﬁsh and rats [87,93,107–109].For example,Hermansky-Pudlak syndrome and amyotrophic lateral sclerosis mis-sense mutations in the Rab38and Fus genes,respectively,were intro-duced in mice [102,110].Subsequently,TALEN-driven HDR was applied to correct the introduced mutation in the Rab38gene [110].TALENs have also been successful in generating human stem cell-based disease models,and restoring expression of functional Dystro-phin in cells from Duchenne muscular dystrophy patients [111,112].New applications include TALEN-mediated generation of a conditional mouse model,human pluripotent stem cell line with conditional transgene expression,knockout of human microRNA genes and single base-editing of an intergenic region upstream of the BUB1gene [113–116].Furthermore,TALE-DNA binding domains enables reversible modulation of mammalian endogenous gene expression and targeted epigenetic chromatin modi ﬁcations [117–119].NH 2Repeat domain COOHTALEN-inducedDSBNon-Homologous End Joining Homology-DirectedRepairA A T T G G C G A NLS ADAL T P E Q V V A I A S N G G G K Q A L E T V Q R L L P V L C Q A H G3411213“Variable di-residue”BA T G C T T T T T T T T C C G A A G G C C G G G C C C C A A A A A G G G G T T T T T C C C A A A A A C T A Fok IFok I5’3’3’5’C G A T T AGene disruptionGene correction Gene additionFig.2.A.Representation of a TALE characterized by an N-terminal domain,a central DNA-binding repeat domain and a C-terminal domain containing two nuclear localization signals (NLS)and the activation domain (AD).A typical repeat is 34amino acids in length and contains two hypervariable amino acids at position 12and 13known as the RVD (highlighted in red).Each RVD is able to recognize one speci ﬁc DNA base pair and serial repeats recognize speci ﬁc DNA sequence and activate expression of speci ﬁc effector host genes through the activation do-main.B.Depiction of a TALEN.A TALEN containing 12speci ﬁc repeats that correspond to binding either thymine (red),cytosine (green),adenine (blue)or guanine (yellow)fused to the nuclease domain of Fok I.Simultaneous binding of a TALEN pair on opposite strands of DNA ﬂanking the target site facilitates dimerization of Fok I and results in a TALEN-induced DSB.Ensuing DNA repair by the NHEJ or HDR pathway can be exploited to introduce the desired genetic modi ﬁcation.1945T.Wijshake et al./Biochimica et Biophysica Acta 1842(2014)1942–1950Similar to ZFNs,a concern of genome editing by TALENs is the occurrence of off-target modiﬁcations.Extensive analysis of known TALEN/DNA cleavage proﬁles determined speciﬁcity scoring of each RVD/nucleotide association,which can be used as a guide in the design of TALENs[120].TALENs can only tolerate limited position-dependent mismatches to keep detectable cleavage activity in vivo,demonstrating its high speciﬁcity[120].In addition,newly synthesized TALEN variants have shown equal on-target cleavage activity and on average ten times lower off-target cleavage activity in human cells[121].Currently,a number of methods have been developed for engineer-ing of TALE repeats[24]:Standard cloning-based[91,92],“Golden Gate”cloning[122–124],iterative capped assembly[125],and the fast ligation-based automatable solid-phase high-throughput(FLASH)sys-tem[126],which has the advantage that construction is rapid,cheap and large number assemblies are feasible.More recent methods include ligation-independent cloning[127]and fairyTALE[128],which enable the high-throughput assembly of TALE repeats.5.Clustered regularly interspaced short palindromic repeats/ CRISPR-associated(CRISPR/Cas)systemIn addition to TALENs and ZFNs,the CRISPR/Cas system has recently been introduced as an efﬁcient and versatile tool for genome editing. CRISPR is an essential part of the immune system of bacteria and ar-chaea directed against foreign nucleic acids[129–131].Upon challenge with a viral or plasmid pathogen,bacteria and archaea integrate short fragments of foreign DNA(protospacers)into their own chromosomes at the proximal end of a repetitive element known as the CRISPR locus/array[129–132].The CRISPR locus is characterized by a series of direct repeats of ap-proximately20–50base pairs separated by unique spacers of similar length[130,131].Transcription of the CRISPR loci into precursor CRISPR RNA(pre-crRNA)is followed by enzymatic cleavage,which re-sults in short crRNAs that can bind with complementary sequences of foreign viruses and plasmids[132–135].Each crRNA is packaged into a surveillance complex to protect the intracellular environment from in-vading viruses and plasmids.crRNA recognizes and mediates the de-struction of foreign DNA sequences through complex formation with CRISPR-associated(Cas)protein that harbors nuclease activity[130–132, 136].Cas proteins are encoded by Cas genes and are localized in the vi-cinity of a CRISPR locus.The cleavage capability of Cas protein9(Cas9) was demonstrated on plasmid DNA containing a protospacer sequence and a protospacer adjacent motif(PAM)sequence,however,the addi-tion of mature crRNA and a trans-activating crRNA(tracrRNA)are es-sential for proper cleavage.TracrRNA enhances crRNA binding to the complementary DNA strand and thereby activates crRNA-guided double-strand DNA(dsDNA)cleavage by Cas9(Fig.3A)(detailed de-scription of the CRISPR/Cas system in bacteria and archaea has been re-ported in[130,132]).Cleavage is site-speciﬁc and occurs3base pairs upstream(arrows in Fig.3A)of the PAM sequence.PAM is a very short stretch of conserved nucleotides in the immediate proximity of the protospacer,and is a determining factor in self versus non-self recogni-tion.Engineering of a crRNA:tracrRNA chimera in the presence of Cas9 was sufﬁcient to cleave a plasmid containing the GFP coding sequence in vitro[132].Heterologous expression of a codon-optimized S.pyogenes Cas9(SpCas9)nuclease,a designed tracrRNA and pre-crRNA have been shown to induce precise RNA-guided cleavage at geno-mic loci in human and mouse cells[25].Similarly,targeted modiﬁcation of loci with SpCas9and a fusion transcript of crRNA-tracrRNA,also known as a guide RNA(gRNA),was successful in human and iPS cells [137,138](Fig.3B).A mutation in the RuvC I domain of SpCas9converts it into a DNA nickase.DNA nickases introduces only a single-strand break or“nick”instead of a DSB,which facilitates homology directed re-pair with highﬁdelity[25,137].CRISPR technology offers the ability to edit different loci simulta-neously or to generate large deletions in mammalian genomes[25,137].Co-injection into zygotes of Tet1and Tet2sgRNA with Cas9 mRNA resulted in mice carrying bi-allelic mutations in both genes with high efﬁciency and speciﬁcity[139].The CRISPR/Cas system has been applied to modify single and/or multiple genes by either NHEJ-or HDR-mediated repair in numerous model organisms,including zebraﬁsh[97,140–143],Drosophila[144–146],rats[147–149],mice [139,147,150–155]and cynomolgus monkeys[156].Furthermore,co-injection into zygotes of Cas9mRNA,various gRNAs and DNA vectors of different sizes encoding a tag orﬂuorescent reporter construct result-ed in mice carrying small insertions or reporter genes.Likewise,using CRISPR/Cas together with a DNA repair template has been successful in creating a conditionalﬂoxed allele in mice[157].Although analysis of potential off-target sites ofﬁve gRNAs in gene-modiﬁed mice and mouse ES cells identiﬁed off-target mutations with low frequency[157],the risk for off-target cleavage by CRISPR/Cas has become an important discussion point,especially since multiple mis-matches are tolerated by the CRISPR/Cas system[158–160].These off-target mutations,even in off-target sites harboring up to5mismatches, were often located within coding genes[158,161].Yet,the accepted mismatch depends on the position of the gRNA-DNA interface,e.g. Cas9-mediated cleavage appears abolished when a single mismatch is present in the last10–12nucleotides near the3’end of the gRNA-target site[25,132].Off-target modiﬁcations have been observed in genes with strong homology.The CRISPR/Cas system targeting the hemoglobinβand CCR5genes revealed signiﬁcant off-target cleavage in the related hemoglobinδand CCR2genes in human cells,respectively [162].Off-target modiﬁcations can be minimized by titration of the Cas9 and gRNA dosage and careful design of the gRNA[159,161].Further-more,both gRNA structure and composition can inﬂuence RNA-guide cleavage and diminish off-target mutagenesis[163].Truncated gRNAs with shorter regions of complementarity can be used to reduce unde-sired off-target modiﬁcations and maintain similar on-target genome editing efﬁciencies[164].Another method makes use of a Cas nickase instead of a nuclease.The advantage of this approach is that single-strand nicks in off-target sites are favorably repaired by the high-ﬁdelity base excision repair pathway[165].Cas nickases directed by a pair of gRNAs targeting opposite strands of a target locus can efﬁciently mediate DSBs while signiﬁcantly reducing off-target activity in human cells[160,163,166,167].In addition,utilization of double nicking has en-abled highly efﬁcient NHEJ-mediated DNA insertion,HDR and genomic microdeletions in human cells and mouse zygotes[167].However,re-cent studies have demonstrated that single monomeric nickases can in-duce unwanted indel mutations as well[160,163,164].Elucidation of the crystal structure of Cas9,either without or with binding to gRNAs and DNA,will enhance the functional understanding of Cas9activity and possibly lead to increased speciﬁcity[168–170].Fortunately,a free web-based application is available to facilitate CRISPR/Cas9-mediated mammalian genome engineering;it allows users to select and validate target sequences and identify potential off-target effects [159,171,172].6.Concluding remarksNuclease technology for genome modiﬁcation has brought a number of major advantages in comparison with conventional gene targeting by homologous recombination in mouse ES cells.The efﬁciency of nuclease-mediated genome editing is signiﬁcantly higher,as demon-strated in a number of model organisms,and cell lines from different species.Nuclease technology can drastically reduce the time line to gen-erate mutant mice.Direct injection of ZFNs,TALEN mRNA or gRNA with Cas9into fertilized mouse oocytes can produce targeted mutations in founder animals with high efﬁcacy.Most founder lines have subse-quently been able to transmit the mutated alleles through the germline to their offspring.These approaches avoid the use of ES cells and the construction of large targeting constructs,which are laborious proce-dures.As demonstrated with the CRISPR/Cas system,multiple genes1946T.Wijshake et al./Biochimica et Biophysica Acta1842(2014)1942–1950。

转基因英语作文

Nevertheless, the ethical implications of transgenic technology cannot be overlooked. The creation of transgenic organisms raises complex moral and philosophical questions regarding the manipulation of nature and the welfare of living beings. As we continue to harness the power of biotechnology, it is essential to consider the potential consequences and establish ethical guidelines to ensure the responsible and sustainable use of transgenic technology.
In conclusion, the development and application of transgenic technology have undoubtedly transformed our lives in numerous ways. While it holds great promise for addressing global challenges in agriculture and medicine, it is imperative to approach this technology with caution and foresight. As future biotechnologists and scientists, it is our responsibility to engage in informed discussions and contribute to the ethical and responsible advancement of transgenic technology for the betterment of humanity.

Intravenous administration of adenoviruses targetingtransforming growth factor

ORIGINAL ARTICLEIntravenous administration of adenoviruses targetingtransforming growth factor beta signaling inhibits established bone metastases in 4T1mouse mammary tumor model in an immunocompetent syngeneic hostZ Zhang 1,4,Z Hu 1,4,5,J Gupta 1,JD Krimmel 1,HM Gerseny 1,AF Berg 1,JS Robbins 1,H Du 2,B Prabhakar 3and P Seth 1We have examined the effect of adenoviruses expressing soluble transforming growth factor receptorII-Fc (sTGF b RIIFc)in a 4T1mouse mammary tumor bone metastasis model using syngeneic BALB/c mice.Infection of 4T1cells with a non-replicating adenovirus,Ad(E1À).sT b RFc,or with two oncolytic adenoviruses,Ad.sT b RFc and TAd.sT b RFc,expressing sTGF b RIIFc (the human TERT promoter drives viral replication in TAd.sT b RFc)produced sTGF b RIIFc protein.Oncolytic adenoviruses produced viral replication and induced cytotoxicity in 4T1cells.4T1cells were resistant to the cytotoxic effects of TGF b -1(up to 10ng ml À1).However,TGF b -1induced the phosphorylation of SMAD2and SMAD3,which were inhibited by co-incubation with sTGF b RIIFc protein.TGF b -1also induced interleukin-11,a well-known osteolytic factor.Intracardiac injection of 4T1-luc2cells produced bone metastases by day 4.Intravenous injection of Ad.sT b RFc (on days 5and 7)followed by bioluminescence imaging (BLI)of mice on days 7,11and 14in tumor-bearing mice indicated inhibition of bone metastasis progression (P o 0.05).X-ray radiography of mice on day 14showed a signiﬁcant reduction of the lesion size by Ad.sT b RFc (P o 0.01)and TAd.sT b RFc (P o 0.05).Replication-deﬁcient virus Ad(E1À).sT b RFc expressing sTGF b RIIFc showed some inhibition of bone metastasis,whereas Ad(E1À).Null was not effective in inhibiting bone metastases.Thus,systemic administration of Ad.sT b RFc and TAd.sT b RFc can inhibit bone metastasis in the 4T1mouse mammary tumor model,and can be developed as potential anti-tumor agents for breast cancer.Cancer Gene Therapy (2012)19,630--636;doi:10.1038/cgt.2012.41;published online 29June 2012Keywords:breast cancer;mouse model;oncolytic adenovirus;systemic delivery;TGF bINTRODUCTIONIn the United States alone,of the nearly 209,000women diagnosed with breast cancer each year,about 43,000die.1A majority of the women develop bone metastases,tumor-induced bone destruction,hypercalcemia and spinal cord compression during the advanced stages of breast cancer,thus seriously compromising the lifestyle of the affected patients.2Currently,there are only limited therapies for bone metastases.Although the two types of drugs---bisphosphonates and denosumab,an anti-body against RANKL (receptor activator of nuclear factor kappa-B ligand)---can inhibit bone resorption,their ability to cure bone metastases remains to be established.3Thus,development of novel therapies to treat breast cancer bone metastasis is a major unmet need in medicine.In the recent years,oncolytic adenoviruses have shown some potential in the treatment of cancer.4--12In an attempt to develop novel therapeutic approaches for bone metastases,our laboratory has developed oncolytic adenoviruses that would kill the cancer cells and simultaneously express a soluble form of transforming growth factor beta (TGF b )receptorII-Fc (sTGF b RIIFc)that can targetTGF b -induced signaling pathways.10--12We chose to target the TGF b pathway because high levels of circulating TGF b protein is a poor prognostic marker in breast cancer patients.13,14Furthermore,aberrant TGF b signaling at the bone metastasis site has been postulated to be a key factor in the progression of breast cancer bone metastases.14--19Therefore,there is a growing interest in developing inhibitors of TGF b signaling for the treatment of various cancer metastases.20--24Using an MDA-MB-231human breast cancer bone metastasis model in immunodeﬁcient mice,we have recently shown that intravenous delivery of oncolytic adenoviruses,Ad.sT b RFc and TAd.sT b RFc,in tumor-bearing mice are effective in inhibiting the established bone metastases.11,12However,before initiating a clinical trial in breast cancer patients,it is important to examine the efﬁcacy of these oncolytic adeno-viruses in an immunocompetent animal model because they have the ability to limit adenoviral replication and thus its efﬁcacy.Keeping that in mind,we have now conducted in vitro and in vivo studies using a mouse mammary 4T1tumor cell model.We report here that infection of 4T1cells with recombinant adenoviruses produced transgene expression,and 4T1cellsReceived 13April 2012;revised 30May 2012;accepted 31May 2012;published online 29June 20121Gene Therapy Program,Department of Medicine,NorthShore Research Institute,Evanston,IL,USA;2Center for Clinical and Research Informatics,NorthShore Research Institute,Evanston,IL,USA (an Afﬁliate of the University of Chicago,Chicago,IL,USA);3Department of Microbiology and Immunology,the University of Illinois at Chicago,Chicago,IL,USA.Correspondence:P Seth,Gene Therapy Program,Department of Medicine,NorthShore Research Institute,an afﬁliate of the University of Chicago,Evanston Hospital,2650Ridge Ave,Room B 652,Evanston,IL 60201.E-mail:pseth@ 4The ﬁrst two authors contributed equally to this work.5Present address:Department of Experimental Hematology,Beijing Institute of Radiation Medicine,Beijing,China.Cancer Gene Therapy (2012)19,630--636&2012Nature America,Inc.All rights reserved 0929-1903/12/cgtsupported adenoviral replication and were killed by oncolytic adenoviruses.Although4T1cells were resistant to TGF b-1-induced cytotoxicity,TGF b was able to activate signaling.More importantly, intracardiac inoculation of4T1cells in BALB/c mice produced bone metastases and osteolytic lesions,and thus is an appropriate pre-clinical model for our purpose.We report here that intravenous injections of Ad.sT b RFc and TAd.sT b RFc inhibited the progression of skeletal metastases in BALB/c mice.Based on ourﬁndings,we believe that oncolytic adenoviruses targeting TGF b pathways can be developed for treating breast cancer bone metastases. MATERIALS AND METHODSCell culture4T1(ATCC,Manassas,VA)mouse mammary tumor cells,4T1-luc2(Caliper life sciences,Hopkinton,MA),MV1Lu(ATCC)mink epithelial cells,and HEK293 (ATCC,Manassas,VA)human embryonic kidney cells were grown in Dulbecco’s modiﬁed Eagle’s medium containing10%bovine calf serum (Invitrogen,Grand Island,NY).Adenoviral vectorsAdenoviral vectors used in these studies are:Ad(E1À).Null,an E1minus replication-deﬁcient adenovirus containing no foreign gene;Ad(E1À).GFP,a replication-deﬁcient adenovirus expressing EGFP protein;Ad(E1À).sT b RFc,a replication-deﬁcient adenovirus expressing sTGF b RIIFc gene;Ad.sT b RFc,an oncolytic adenovirus expressing sTGF b RIIFc gene(constructed using dl01/07 mutant of Ad5,containing two deletions in E1A region as previously described)10and TAd.sT b RFc,an oncolytic adenovirus expressing sTGF b RIIFc gene with the human TERT promoter driving the adenoviral replication as published.25Adenoviral vectors were grown in HEK293cells and puriﬁed by double CsCl2gradient as described.26Viral particle(VP)numbers were determined by measuring OD260(optical density260)of the sodium dodecyl sulfate-treated adenoviral solutions.Adenoviral replication assay4T1cells were plated in24-well dishes(2Â105cells/well).The following day,cells were infected with viral vectors(5Â103VPs/cell)and the incubations were continued for3days.Cells were then subjected to immunohistochemistry for adenoviral hexon staining using an Adenoviral Titer commercial kit(Clontech,Mountain view,CA)as described earlier.25,27 Positive hexon expressing brown cells were photographed,and counted under the microscope to quantify viral replication.Cytotoxicity assaysTo measure TGF b-1-induced cytotoxicity,cells were plated in96-well plates 103cells/well.The following day,cells were infected with various concentrations of TGF b-1(0.001--10ng mlÀ1)(Sigma,St Louis,MO),and the incubations were continued for7days.Cells were washed,ﬁxed and stained with sulforhodamine B(Sigma),and the A564(absorbance at564nm) measured as previously described.26Untreated control cells were considered to have100%survival.To examine viral-induced cytotoxicity,the same protocol was used except that4T1cells were incubated with various doses of adenoviral vectors for7days before sulforhodamine B staining.GFP expression4T1cells were plated in6-well dishes(4Â105cells/well).The following day, cells were infected with Ad(E1À).GFP(2.5Â104VPs/cell)and incubated for 48h.Cells were photographed using aﬂuorescent microscope(Â200). sTGF b RIIFc expressionTo examine adenoviral vector-mediated sTGF b RIIFc expression,4T1cells were plated in6-well dishes(4Â105cells/well).The following day,cells were infected with various viral vectors(2.5Â104VPs/cell).After24h, media was changed to serum-free media,and the incubations continued for another24h.sTGF b RIIFc expression in the media and the cell lysates were examined by western blot analyses as previously described.10sTGF b RIIFc protein amounts in the media were measured by enzyme-linked immunosorbent assay using antibodies against the human IgG Fc fragment (Jackson ImmunoResearch,West Grove,PA,USA)as previously described.11SMAD phosphorylation4T1cells were plated in6-well plates(4Â105cells/well).The following day,cells were serum starved for6h,and then treated with TGF b-1(1ng mlÀ1)in the absence or presence of sTGF b RIIFc(250ng mlÀ1)for1h.Cells were analyzed for p-SMAD2,p-SMAD3and for total SMAD2/3using westernblots as previously described.28The blots were visualized by enhanced chemiluminescence substrate(Amersham Biosciences,Piscataway,NJ).Interleukin(IL)-11assays4T1cells were plated in6-well plates(2Â105cells per well).The following day,cells were serum starved over night,and then exposed to TGF b-1(0.1,Phase80. 440. 7Kda80. 440. 7A d.s TR FcT Ad.sT RF c40. 7A d(E1-).s TR FcA d(E1-).Nu l lM oc ksTGF RIIFc(Media)sTGF RIIFc(Lysate)Actin(Lysate)MockAd((E1-).NullAd(E1-).sTRFcAd.sTRFcTAd.sTRFc5101520sTGFRIIFc(g/ml)FluorescenceFigure1.Adenoviral-mediated transgene expression in4T1cells.(a)4T1cells were infected with Ad(E1À).GFP(2.5Â104VPs/cell)for24h. Cells were photographed(Â200)using aﬂuorescent microscope. Same viewingﬁelds were used to take phase contrast(left)orﬂuorescent(right)images.(b)4T1cells were infected with various adenoviral vectors(2.5Â104VPs/cell).Cell lysates and media were analyzed by western blots for sTGF b RIIFc protein expression.(c) Extracellular media were used to examine sTGF b RIIFC levels by en-zyme-linked immunosorbent assay method.Adenoviruses targeting TGF b for bone metastasisZ Zhang et al631Cancer Gene Therapy(2012),630--636&2012Nature America,Inc.1or 5ng ml À1)for 48h.Media were analyzed for IL-11levels by enzyme-linked immunosorbent assay using the previously described method.28Animal modelAll animal experiments were conducted using the animal protocols approved by the IACUC committee of the NorthShore University HealthSystem.To establish bone metastases,4T1-luc2cells were injected in the left heart ventricle (day 0)of 4-week-old BALB/c mice (Charles River laboratories,Wilmington,MA).On day 4,the mice were subjected to bioluminescence imaging (BLI)in dorsal and ventral positions using Xenogen IVIS Spectrum imaging equipment (Caliper life sciences,Hopkinton,MA).Photon signals were quantiﬁed using living image software 3.0(Caliper life sciences,Hopkinton,MA)as previously described.12Mice were divided into various groups,with statistically indistinguishable BLI signals among each group.Various viral vectors were administered via tail vein on days 5and 7(5Â1010VPs per injection/mouse,each injection in a 0.1-ml volume).The control group of mice was administered the buffer alone.BLIMice were imaged in dorsal and ventral positions on days 7,11and 14using the IVIS Spectrum imaging system (Caliper Life Sciences).Whole-body BLI signals were used to quantify the metastasis as previouslydescribed.12Signals in the hind limbs were separately quantiﬁed to measure the skeletal metastases as described.12X-ray radiographyOn day 14following tumor cell injections,mice were also subjected to X-ray radiography in prone position using Faxitron (Faxitron X-ray Corpora-tion,Wheeling,IL).Skeletal lesion sizes were measured in the femur and tibia of both the hind limbs using Image J software as described earlier.11,12Statistical evaluationAll statistical analyses were performed using GraphPad Prizm 5(GraphPad software,San Diego,CA).Data are presented as mean ±s.e.m.To analyze BLI signal progression,a two-way repeated-measure analysis of variance followed by Bonferroni post-tests was used.For multiple groups,statistical signiﬁcance was analyzed using one-way analysis of variancefollowed by Bonferroni post-tests.P o 0.05was considered a statistically signiﬁcant difference.RESULTS4T1cells can be infected with human adenoviral vectorsExperiments were conducted to examine the infectability of 4T1cells with replication-deﬁcient and replication-competent adenoviral vectors.4T1cells were infected with Ad(E1À).GFP,aFigure 2.Adenoviral replication and cytotoxicity in 4T1cells.(a )4T1cells were infected with various viral vectors for 72h,and stained for hexon protein.(b )Hexon-positive cells were counted in each well to measure the viral titers.(c )The ratios between the viral titer of each virus and that of Ad(E1À).Null are shown.(d )4T1cells were exposed to various viral vectors for 7days.The cytotoxicity assays were conducted by sulforhodamine B staining.Control cells were considered to have 100%survival.(e )IC 50(half maximal inhibitory concentration)for each virus was calculated,and the IC 50ratios between each vector and Ad(E1À).Null are shown.***P o 0.001,**P o 0.01.Adenoviruses targeting TGF b for bone metastasisZ Zhang et al632Cancer Gene Therapy (2012),630--636&2012Nature America,Inc.non-replicating adenovirus,for48h,and the cells were visualized under aﬂuorescent microscope.The vast majority of cells produced a strong GFP signal(Figure1a).In another experiment,cells were infected with Ad(E1À).sT b RFc,a replication-deﬁcient adenovirus, and two oncolytic adenoviruses---Ad.sT b RFc and TAd.sT b RFc.Cell lysates and the extracellular media were subjected to western blot analyses for sTGF b RIIFc expression.Infection of4T1cells with Ad(E1À).sT b RFc,Ad.sT b RFc and TAd.sT b RFc resulted in sTGF b RIIFc protein production,which could be detected in the cell lysates as well as the extracellular media(Figure1b).The amounts of sTGF b RIIFc were quantiﬁed in the media using enzyme-linked immunosorbent assay,and were found to be in the range of 6.21--15.48m g mlÀ1of media(Figure1c).These results indicate that4T1cells can be infected with human adenoviruses and that infection with Ad(E1À).sT b RFc,Ad.sT b RFc or TAd.sT b RFc leads to the production of sTGF b RIIFc protein,which is secreted into the media.Oncolytic adenoviruses replicate and induce cytotoxicity in4T1 cellsNext,we examined the replication potential of adenoviral vectors in4T1cells.Cells were incubated with various adenoviral vectors (5Â103VPs/cell)at371C for72h,and viral titer was determined by hexon staining.Figure2a shows typical hexon staining of4T1 cells exposed to various viral vectors.There were very few hexon expressing brown cells in Ad(E1À).Null-or Ad(E1À).sT b RFc-treated samples(Figure2a).However,a large number of4T1cells infected with Ad.sT b RFc or TAd.sT b RFc were hexon positive(Figure2a). Quantiﬁcation of hexon-positive cells indicated that Ad.sT b RFc produced viral titers(Figures2b and c),which were about257-times higher than the non-replicating adenovirus Ad(E1À).Null (P o0.001).TAd.sT b RFc produced175-times higher viral titer than Ad(E1À).Null(P o0.01)(Figures2b and c).However,viral titers in Ad(E1À).sT b RFc-infected cells were similar to those in Ad(E1À).Null-treated cells(Figures2b and c).From these results, we conclude that continuous incubation of4T1cells with oncolytic adenoviruses can produce viral replication.To examine whether viral replication can result in cytotoxicity, 4T1cells were incubated with various adenoviral vectors for 7days,and the cytotoxicity assays were performed.Both the oncolytic adenoviruses,Ad.sT b RFc and TAd.sT b RFc,produced a dose-dependent cytotoxicity in4T1cells(Figure2d).Based on the IC50values,Ad.sT b RFc and TAd.sT b RFc were about34.2-fold and 24.0-fold,respectively,more toxic than the non-replicating virus Ad(E1À).Null(Figure2e).By contrast,a non-replicating virus Ad(E1À).sT b RFc produced toxicity that was comparable with Ad(E1À).Null(Figures2d and e).4T1cells are resistant to killing by TGF b,but retain TGF b-mediated signaling pathwaysNext,we investigated the killing effect of TGF b in4T1cells.4T1 cells were exposed to various concentrations of TGF b-1,and7days later cytotoxicity was measured.As a positive control, another rodent cell-type MV1Lu,known to be sensitive to TGF b, was used.As shown in Figure3a,there was little to no cytotoxic effect of TGF b-1even at the highest concentration used(10ng mlÀ1)in4T1cells.However,MV1Lu cells were killed even by a very low concentration of TGF b-1,with an IC50of o0.1ng mlÀ1.To examine whether TGF b could induce signaling in4T1cells, we exposed4T1cells to TGF b-1and analyzed the cell lysates for SMAD2and SMAD3phosphorylation.Figure3b shows that TGF b-1 induced SMAD2and SMAD3phosphorylation in4T1cells.Co-incubation of sTGF b RIIFc with TGF b-1inhibited TGF b-1-dependent SMAD2and SMAD3phosphorylation(Figure3b).We also examined the effect of TGF b-1on IL-11production,a known osteolytic factor in human breast cancer cells.28,29TGF b-1induced IL-11protein production in a dose-dependent manner(Figure3c).These results indicate that4T1cells respond to TGF b-1and undergo activation of signaling pathways that are known to favor bone metastases in human breast cancer cells.18,28,30Importantly, sTGF b RIIFc is able to abolish the TGF b signaling.Oncolytic adenoviral-mediated inhibition of4T1-induced metastases:BLI analysesNext,we examined the effect of systemic administration of adenoviral vectors expressing sTGFRIIFc in a4T1bone metastasis model.4T1-luc2cells were inoculated into the left heart ventriclesof BALB/c mice.After4days,mice were subjected to whole-bodyBLI in both dorsal and ventral positions.Mice were split into multiple groups,with nearly equal BLI signal within each group.Two doses of adenoviral vectors were given via the tailvein---theTGF-++---++sTGF RIIFcp-SMAD2p-SMAD3SMAD2/3501001500.001TGF -1 (ng/ml)%Survival0.1 1.0 5.00.00.30.60.91.2TGF -1 (ng/ml)IL-11(ng/ml)10.01.00.10.01Figure3.Effect of TGF b-1on4T1cells.(a)4T1or MV1Lu cells were exposed to various concentrations of TGF b-1.After7days,cyto-toxicity assays were conducted using sulforhodamine B staining. Control cells were considered to have100%survival.(b)4T1cells were exposed to TGF b-1(1ng mlÀ1)for60min in the absence or presence of sTGF b RIIFc(250ng mlÀ1).Cell lysates were examined forp-SMAD2,p-SMAD3and total SMAD2/3by western blot analysis.(c)4T1cells were exposed to various concentrations of TGF b-1for48h.Cell media were used to measure IL-11levels by enzyme-linked immunosorbent assay method.Adenoviruses targeting TGF b for bone metastasisZ Zhang et al633Cancer Gene Therapy(2012),630--636&2012Nature America,Inc.ﬁrst dose on day 5(5Â1010VPs/mouse)and a second dose on day 7(5Â1010VPs/mouse).Mice were subjected to BLI on days 7,11and 14following tumor-cell injection.A representative mouse showing BLI signal from each treatment group is shown in Figure 4a.Whole-body BLI signals were quantiﬁed and are shown in Figure 4b.There was a time-dependent increase in the whole-body BLI signal to 0.88Â1011photons sec À1in the control group of mice that received buffer alone (Figure 4b).There was no signiﬁcant inhibition of BLI signal in the Ad(E1À).Null,Ad(E1À).sT b RFc or TAd.sT b RFc treatment groups (Figure 4b).However,Ad.sT b RFc induced a signiﬁcant inhibition (P o 0.05)of the whole-body BLI.As 4T1cells also established bone metastasis in the hind limbs (Figure 4a),the effect of viral vectors on the BLI signal in the hind limbs was quantiﬁed.In the control group of mice,the BLI signal in the hind limbs reached to 0.86Â1010photons sec À1,and there was a signiﬁcant inhibition of BLI signal accumulation in the hind limbs in the Ad.sT b RFc-treated group (P o 0.05).However,Ad(E1À).Null,Ad(E1À).sT b RFc and TAd.sT b RFc treatments had no signiﬁcant effect on the BLI signal intensity in the hind limbs (Figure 4c).Oncolytic adenoviral vectors’-mediated inhibition of 4T1-induced metastases:X-ray analysesTo further examine the effects of vectors’administration on bone metastases,mice were subjected to X-ray radiography on day 14.A representative example of X-ray radiographs from each group is shown in Figure 5a.The long bones in buffer-treated and Ad(E1À).Null-treated mice had large osteolytic lesions,as in-dicated by red arrows.The lesion sizes were relatively smaller in the other treatment groups.The tumor lesions in the hind limbs were quantiﬁed using the Image J program and are shown in Figure 5b.Tumor sizes in the buffer group were 6.92±1.27mm 2.Tumor sizes in Ad(E1À).Null,Ad(E1À).sT b RFc,Ad.sT b RFc and TAd.sT b RFc treatment groups were 5.79±0.86, 3.56±0.72,2.30±0.69and 2.94±0.57mm 2,respectively.These results indicate that although Ad(E1À).Null and Ad(E1À).sT b RFc had no signiﬁcant effect on the tumor sizes,a signiﬁcant inhibition of the tumor growth was observed in the Ad.sT b RFc (P o 0.01)and TAd.sT b RFc (P o 0.05)treatment groups (Figure 5b).Although we were able to monitor BLI and X-ray until day 14,by day 18a number of animal deaths were observed in each of the treatment groups.The following ratios indicate the number of mice that died between days 14and 18over the initial number of mice in each group:buffer,3:9;Ad(E1À).Null,4:9;Ad(E1À).sT b RFc,3:9;Ad.sT b RFc,2:11;and TAd.sT b RFc,3:11.To conﬁrm Ad(E1À).sT b RFc-,Ad.sT b RFc-and TAd.sT b RFc-mediated sTGF b RIIFc expression in the blood,samples were collected from the remaining mice on day 18and the sTGF b RIIFc production was analyzed by enzyme-linked immunosorbent assay.The results indicated high levels:2.43±1.67,6.53±16.31and 15.41±24.86m g ml À1of sTGF b RIIFc in the blood samples from theA d .s T R F c TA d .s TR F c A d(E 1-).N u l l A d.(E 1-).s TRF cB uf f erDays Post-CellsInjection W h o l e B o d yP h o t o n s /s e c (x 10-11)D a y 4D a y 7D a y 11D a y 14471114Days Post-CellsInjectionH i n d L i m b s P h o t o n s /s e c (10-10)Figure 4.Effect of systemic delivery of viral vectors on 4T1bone metastases:BLI analysis.4T1-luc2cells were injected in BALB/c mice (5Â104cells/mouse)on day 0.Initial BLI was performed on day 4;mice with positive tumors were administered viral vectors or buffer (via tail vein)on days 5and 7.(a)BLI was conducted on days 7,11and 14.The numbers of mice in each treatment group were:buffer (n ¼9),Ad(E1À).Null (n ¼9),Ad(E1À).sT b RFc (n ¼9),Ad.sT b RFc (n ¼11)and TAd.sT b RFc (n ¼11).Representative mice of each treatment group are shown.(b )BLI signal in the whole body of mice in various treatment groups were quantiﬁed and are shown.(c )To measure bone metastases,BLI signals in the hind limbs (shown by red circles)were quantiﬁed in each treatment group and are shown.*P o 0.05.Adenoviruses targeting TGF b for bone metastasisZ Zhang et al634Cancer Gene Therapy (2012),630--636&2012Nature America,Inc.Ad(E1À).sT b RFc,Ad.sT b RFc and TAd.sT b RFc treatment groups, respectively.Thus,although the intravenous injection of Ad(E1À).sT b RFc,Ad.sT b RFc and TAd.sT b RFc resulted in sTGF b RIIFc production,it appears that the replicating viruses expressing sTGF b RIIFc were the most effective in inhibiting bone metastases. DISCUSSIONThe keyﬁnding here is that intravenous delivery of oncolytic virus Ad.sT b RFc expressing sTGF b RIIFc can inhibit bone metastasis in the4T1mouse mammary tumor bone metastasis model in a syngeneic host as revealed by BLI studies.X-ray radiographic analyses showed inhibition of tumor growth by Ad.sT b RFc and TAd.sT b RFc,though Ad.sT b RFc was superior to TAd.sT b RFc.A non-replicating virus,Ad(E1À).sT b RFc,expressing sTGF b RIIFc showed some inhibition of bone metastasis in X-ray analyses;Ad(E1À).Null was not effective in either BLI or X-ray analyses.Most of the previously published studies using oncolytic adenoviruses have been conducted in human xenografts estab-lished in immunodeﬁcient nude mice,mainly because mouse tumor cells are not considered good targets for the human adenoviruses.However,it is critical that we continue to explore the animal models in which oncolytic adenoviruses can be examined in immunocompetent syngeneic hosts as described here.It is quite interesting that4T1cells can be infected with human adenoviruses resulting in high levels of transgene expression,indicating the presence of adenoviral receptors even in mouse4T1cells.Moreover,continuous exposure of4T1cells to oncolytic adenoviral vectors can produce a viral titer.This indicates that human adenoviruses can result in virus entry and replication,clearly demonstrating that human adenoviruses can indeed infect and replicate in4T1mouse tumor cells,which is consistent with a previous report.31This infection could be via the previously known adenoviral receptor,by an unknown adenoviral receptor or even by other pathways,including clathrin-indepen-dent mechanisms such as macropinocytosis,phagocytosis or trans-endocytosis.32Once the VPs are internalized by the cells, however,viral replication proceeds as in human breast cancer cells.Although the exact relationship of the Ad.sT b RFc-and TAd.sT b RFc-induced replication resulting in the cytotoxicity of the 4T1tumor model remains to be examined,it is tempting to speculate that the viral replication resulting in the cytotoxic effects of the adenoviral vectors in the mouse tumor cells could have a role in mediating the in vivo anti-tumor responses reported here.Another important observation is the inability of TGF b to kill4T1 cells and yet induce the TGF b signaling pathway(SMAD-phosphorylation),and the production of IL-11(a well-known osteolytic factor in human breast cancer bone metastasis).Thus,in this regard,4T1is an appropriate tumor model for examining the role of TGF b signaling in bone metastases.In the radiographic analyses,a non-replicating adenovirus expressing sTGF b RIIFc showed some inhibition of bone metastasis,albeit weaker than oncolytic adenovirus Ad.sT b RFc.Again,these studies suggest that the expression of sTGF b RIIFc,coupled with viral replication and cytotoxicity,is potentially having a role in mediating the inhibition of bone metastases.Intravenous delivery of adenoviruses will result in their uptake mainly in the liver,and in smaller amounts in other tissues and the skeletal tumors.11,12,27,33We believe that the infection of tumor cells in vivo will result in the viral replication in the tumor cells causing cell killing and partial tumor destruction.Infection of tumor cells and other mouse organs will result in the production of sTGF b RIIFc that will be released in the blood.The sTGF b RIIFc production resulting in the inhibition of TGF b signaling at the tumor/bone site will also contribute towards the inhibition of bone metastases.Among the three vectors expressing sTGF b RIIFc, the most effective vector is the Ad.sT b RFc;TAd.sT b RFc is slightly weaker than Ad.sT b RFc,and the least effective is the Ad(E1À).sT b RFc.As all the three vectors produce nearly equal amounts of sTGF b RIIFc,Ad.sT b RFc is the most effective,which is perhaps due to its higher replication potential in the tumor cells. TAd.sT b RFc can also replicate in the tumor cells,but its replication potential is slightly lower than Ad.sT b RFc;and Ad(E1À).sT b RFc is replication-deﬁcient.Based on these results,we believe that both viral replication and the sTGF b RIIFc expression have an important role in the inhibition of bone metastases.In addition to understanding the role of viral replication and the inhibition of TGF b signaling at the tumor--bone microenviron-ment,the future availability of a4T1bone metastasis model will also allow us to further explore the role of the adenoviral vector-induced innate and humoral immune responses,34--36the role of TGF b in suppressing the immune system14,15,37and how that can be reversed by the oncolytic adenoviruses expressing sTGF b RIIFc. These questions can be addressed only in fully immunocompetent animal models.In conclusion,our work described here shows that oncolytic adenoviruses targeting the TGF b pathway can inhibit breast cancer bone metastases in a mouse mammary tumor model established in a syngeneic immunocompetent host and repre-sents an important step in developing oncolytic adenoviruses for the treatment of breast cancer bone metastases.This animal model will now allow us to investigate the underlying molecular mechanism of action of the oncolytic adenoviruses,which may help in reﬁning this method of treatment.CONFLICT OF INTERESTThe authors declare no conﬂict of interest.B u f f e rA d(E1-).s TR F cT A d.s TR F cA d.s T RF cA d(E1-).N ul lBufferAd(E1-).NullAd(E1-).sTRFcAd.sTRFcTAd.sTRFc246810Tumorarea(mm2)Figure5.Effect of systemic delivery of viral vectors on4T1bone metastases:X-ray radiography.(a)Mice from the above experiment described in Figure4,were subjected to X-ray radiography on day 14.(b)Lesion sizes in each mouse were calculated using Image J software.Results shown are the average lesion sizes in the hind limbs in each of the treatment groups.The numbers of mice in each treatment group were:buffer(n¼9),Ad(E1À).Null(n¼9), Ad(E1À).sT b RFc(n¼9),Ad.sT b RFc(n¼11)and TAd.sT b RFc(n¼11). *P o0.05,**P o0.01.Adenoviruses targeting TGF b for bone metastasisZ Zhang et al635Cancer Gene Therapy(2012),630--636&2012Nature America,Inc.。

Microbial Biocontrol Agent Sample

Microbial Biocontrol Agent Sample As a microbial biocontrol agent sample, I understand the importance of my role in controlling harmful pests and diseases in various agricultural and environmental settings. My purpose is to provide an alternative to chemical pesticides and fungicides, offering a more sustainable and environmentallyfriendly solution to pest and disease management. However, there are various challenges and considerations that need to be addressed when it comes to the useof microbial biocontrol agents, and it is important to explore these perspectivesin order to fully understand the impact and potential of this approach. One perspective to consider is the effectiveness of microbial biocontrol agents in comparison to traditional chemical pesticides. While chemical pesticides have been widely used for pest and disease management, there is growing concern over their negative impact on human health and the environment. Microbial biocontrol agents offer a promising alternative, as they can effectively control pests and diseases without the harmful side effects associated with chemical pesticides. Research has shown that microbial biocontrol agents can be just as effective, if not more so, than chemical pesticides in certain situations, making them a valuable tool in sustainable agriculture and environmental management. Another important perspective to consider is the safety and regulatory aspects of using microbial biocontrol agents. It is crucial to ensure that these agents are safe for humans, animals, and the environment, and that they do not pose any unintended risks. Regulatory agencies play a key role in evaluating and approving microbialbiocontrol agents for use, and it is essential to adhere to their guidelines and requirements to ensure the safety and efficacy of these products. Additionally, proper education and training on the use of microbial biocontrol agents is important to ensure that they are used responsibly and effectively. Furthermore, the economic perspective of using microbial biocontrol agents is worth considering. While chemical pesticides may seem more cost-effective in the short term, thelong-term benefits of using microbial biocontrol agents should not be overlooked. By reducing the reliance on chemical pesticides, microbial biocontrol agents can contribute to improved soil health, reduced chemical residues in food and the environment, and overall sustainability of agricultural and environmentalpractices. It is important to consider the potential economic benefits of using microbial biocontrol agents in the long term, as well as the potential cost savings associated with reduced chemical pesticide use. In addition to these perspectives, it is important to acknowledge the challenges and limitations associated with the use of microbial biocontrol agents. One challenge is the variability in effectiveness of different microbial strains, as well as their susceptibility to environmental factors. It is important to conduct thorough research and testing to identify the most effective microbial biocontrol agentsfor specific pests and diseases, as well as the best application methods for different environmental conditions. Additionally, there may be limitations in the availability and accessibility of microbial biocontrol agents, particularly in developing countries or remote areas. Efforts should be made to address these challenges and ensure that microbial biocontrol agents are widely accessible and applicable in diverse agricultural and environmental settings. Overall, the use of microbial biocontrol agents presents a promising and sustainable approach to pest and disease management in agriculture and environmental settings. By considering the various perspectives and addressing the challenges and limitations associated with their use, we can work towards harnessing the full potential of microbial biocontrol agents and promoting their widespread adoption. It is important to continue research and development in this field, as well as to educate and empower stakeholders to make informed decisions about the use of microbial biocontrol agents. Together, we can work towards a more sustainable and environmentally friendly approach to pest and disease management, ensuring the health and well-being of our ecosystems and communities.。

Molecular Devices QPix 400 Series Microbial Colony

What is synthetic biology?Synthetic biology is a broad term that refers to the manipulation of genetic pathways to harness the power of existing biological systems in novel ways (often to manufacture molecules or proteins). Synthetic biology applies principles that are derived from engineering, specifically design-build-test-learn cycles, to biological systems. By leveraging high-throughput workflows, synthetic biologists can accelerate this process.Colony picking in synthetic biology©2021 Molecular Devices, LLC. The trademarks used herein are the property of Molecular Devices, LLC or their respective owners. Specifications subject to change without notice. Patents: /productpatents FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. QPix™ 400 Series Microbial Colony PickersThe QPix picker can be integrated with other lab components such as incubators, liquid handlers, and robotics for a fully automated work cell. Our customization and automation team can tailor QPix colony pickers and deploy the integration, or provide an open API and software support for the integration process.Benefits of automated colony picking• E nables higher throughput while minimizing manual laborCopiesReplicatingSelected coloniesRe-arrayingConsolidate best clonesthe use of multiple formats of source and destination plates Objective software data analysis allow Acoustic sensors detectagar height, helping high-precision robotics to pick single colonies gently and accurately Organism-specific, handle multiple organisms Wash baths and halogen heat sterilization。

引导编辑系统研究进展

华南农业大学学报 Journal of South China Agricultural University 2024, 45(2): 159-171DOI: 10.7671/j.issn.1001-411X.202309002林秋鹏, 朱秀丽, 马琳莎, 等. 引导编辑系统研究进展[J]. 华南农业大学学报, 2024, 45(2): 159-171.LIN Qiupeng, ZHU Xiuli, MA Linsha, et al. Recent advances in prime editing system[J]. Journal of South China Agricultural University, 2024, 45(2): 159-171.特约综述引导编辑系统研究进展林秋鹏†，朱秀丽†，马琳莎，姚鹏程(广东省植物分子育种重点实验室/华南农业大学农学院, 广东广州 510642)摘要: 引导编辑(Prime editing，PE)系统是一种全新的、革命性的基因组编辑策略。

该系统由引导编辑器(Primeeditor)组成，包括nCas9(H840A)与逆转录酶(Reverse transcriptase，RT)的融合蛋白；以及包含PBS(Primerbinding site)序列和RT模板(RT template，RTT)序列的pegRNA(Prime editing guide RNA)两大部分。

PE系统可以在双链不断裂的情况下实现所有12种类型的碱基替换及小片段DNA增删，是精准编辑的全新范式。

自2019年开发至今不到4年时间，PE系统作为一种通用的技术平台，已广泛应用于医疗、农业等各个领域，产生了一大批新种质资源、基因治疗药物等优秀应用案例。

PE作为目前最灵活、最具发展前景的基因组精准编辑新手段，仍旧存在效率偏低、大片段操纵能力不足、系统组分设计复杂(如pegRNA)、安全性未全面评估等问题，仍需要深入研究。

英语转基因作文

英语转基因作文Title: The Controversy Surrounding Genetically Modified Organisms (GMOs)。

Genetically modified organisms (GMOs) have been a topic of heated debate for decades. While proponents argue for their potential to address global food security challenges, opponents raise concerns about their safety and environmental impact. This essay delves into the various perspectives surrounding GMOs.To begin with, proponents of GMOs emphasize their potential benefits, particularly in addressing foodscarcity issues. By genetically modifying crops, scientists can enhance their resistance to pests, diseases, and environmental stressors, thereby increasing yields and ensuring food security for a growing global population. Additionally, GMOs can be engineered to possess desirable traits such as drought tolerance and nutritional enhancements, potentially improving the nutritional valueof food crops.Moreover, proponents argue that GMOs offer environmental benefits by reducing the need for chemical pesticides and herbicides. Through the development of insect-resistant and herbicide-tolerant crops, farmers can minimize the use of harmful agrochemicals, thereby mitigating environmental damage and promoting sustainable agriculture practices.On the other hand, opponents of GMOs raise significant concerns regarding their safety and long-term impact on human health and the environment. One of the primary concerns is the potential for unintended consequences and unforeseen risks associated with genetic modification. Critics argue that altering the genetic makeup of organisms in such a fundamental way may have unpredictable consequences, including the creation of new allergens or toxins.Furthermore, opponents highlight the monopolistic control of the seed market by biotechnology corporations asa major ethical concern. The widespread adoption of GMOs has led to the consolidation of seed companies, limiting farmers' choices and increasing their dependency on corporate-controlled agricultural technologies. This concentration of power raises questions about food sovereignty and the equitable distribution of resources in the agricultural sector.In addition, environmental activists express concerns about the potential ecological impacts of GMOs, including the contamination of native species through cross-pollination and the disruption of ecosystems. The introduction of genetically modified organisms into the environment may have unintended consequences, such as the emergence of superweeds or the decline of beneficial insect populations.In conclusion, the debate surrounding genetically modified organisms remains contentious, with proponents advocating for their potential to address global food security challenges and opponents raising concerns about their safety and environmental impact. As advancements inbiotechnology continue to accelerate, it is essential to consider the ethical, social, and environmental implications of GMOs and engage in informed dialogue to navigate this complex issue responsibly.。

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Turning Control of a Multilink Biomimetic Robotic Fish
Junzhi Yu, Lizhong Liu, Long Wang, Min Tan, and De Xu
Abstract—This paper deals with maneuver issues of a multilink biomimetic robotic ﬁsh, particularly focusing on turning control in free swimming. The characteristic parameters determining turning performance involve magnitude, position, and time of the deﬂections applied to the links, which are discussed via a series of simulation calculations and actual experiments. Index Terms—Biomimetic robotic ﬁsh, maneuver, path planning, turning control, underwater robot.
as course corrections due to external perturbations [1]. To adapt 3-D complex corals, tropic ﬁsh are found to acquire high maneuverability with turning radii of <0.06 BL, where BL is the total body length. Data also indicate that ﬁshes ranging in length from 0.04 to 0.39 m are able to turn at rates of 425.6–7300.6◦ /s [2]. A conventional unmanned undersea vehicle (UUV); however, executes a turn at approximately 3– 5◦ /s, often requiring several body lengths as its turning radius. Inspired by extraordinary performance in propulsion and low speed, precise maneuverability of ﬁsh, both scientists and engineers have shown an increased interest in building biorobotic autonomous undersea vehicles to endow them with ﬁsh-like functional and operational capabilities [3]–[6]. For more detailed background discussion on the ﬁsh-like swimming robot, i.e., robotic ﬁsh, refer to the latest literature [7] and [8]. In the context of biology, maneuver addressed by biologists usually refers to turning. To date, little is known about the biological mechanisms governing complex maneuvers of ﬁshes except fast-starts involving C-shaped and S-shaped maneuver. Theoretical and experimental efforts in creating ﬁsh-like robots as agile as a real ﬁsh have been undertaken for years; however, the results are not very satisfactory: only a part of maneuverability has been replicated, and corresponding maneuver performance is markedly degraded at higher speeds. Among the noteworthy work, Bandyopadhyay [9] and Kato [10] studied the use of ﬁns in small underwater vehicles for hovering and low-speed maneuvering. Read et al. [11] explored the possibility of oscillating foils for propulsion and maneuvering. McIsaac and Ostrowski [12] developed theoretical justiﬁcation for a turning gait of anguilliform locomotion. Through varying frequency, amplitude, and leaning of the tail, Hirata et al. [13] experimentally discussed turning modes of a two-joint ﬁsh robot that uses tail swing. Thereafter, Liu and Hu [14] kinematically discussed the C-shaped turning of a robotic ﬁsh with a four-DOF tail. To replicate the force/elongation of natural muscles, Stefanini et al. [15] developed a combined actuator applied to a lamprey-like robot. Furthermore, Tsakiris et al. [16] and Sfakiotakis and Tsakiris [17] established and veriﬁed computational models of central-pattern-generator-based locomotion control for undulatory robotic systems, where forward and turning undulatory gaits were achieved by varying the tonic inputs. The objective of this paper, on the basis of our previous research on the robotic ﬁsh [7], [18], [19], is to extend Hirata’s turning control method to a more general case, with emphasis on both executing various turns agilely and operating conveniently. Compared with turns achieved by real ﬁsh due primarily to the use of multiple propulsors, we add different deﬂections (i.e., dynamic offsets) to the straight, symmetric swimming gaits propelled only by an oscillating posterior body and tail ﬁn to accomplish various turns, which are easily handled in engineering practice. II. DEVELOPMENT OF BIOMIMETIC ROBOTIC FISH In this section, we brieﬂy present an overall design of a multilink biomimetic ﬁsh prototype, describing its propulsive mechanism and mechatronic design. A. Multilink-Based Propulsive Conﬁguration As illustrated in Fig. 1, the propulsive structure treated here is a free-swimming, multilink robotic ﬁsh. It is composed of three parts: an anterior body with a pair of pectoral ﬁns, a ﬂexible multilink body, and an oscillating lunate caudal ﬁn. The ﬂexible multilink body is supposed with N links and N − 1 revolute-type joints independently actuated by motors. Since the ﬁsh tail moving in a straight cruise is generally described as a posteriorly propagating wave (1) [20], the desired motion for the multilink is expected to approximate the so-called body wave