Gene targeting technology is an important means to investigate gene functions, but its efficiency of gene targeting is very low, especially for somatic cell targeting. Artificially induced double-strand breaks （DSB）...Gene targeting technology is an important means to investigate gene functions, but its efficiency of gene targeting is very low, especially for somatic cell targeting. Artificially induced double-strand breaks （DSB） and triplex forming oligonucleotide （TFO） are currently developed methods to improve the targeting efficiency. This paper summarized the basic principles, design ideas and application in gene targeting efficiency improvement of these two methods, analyzed and com- pared their characteristics, and finally proposed prospects for their future development.展开更多
The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer v...The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL 1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.展开更多
Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors （TALEs） contain a DNA binding domain consist...Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors （TALEs） contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases （TALENs）, constructed using a method called ＂unit assembly＂, specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non- homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.展开更多
Targeted genome modifications with the Cas9/gRNA system derived from the clustered regularly interspaced short palin- dromic repeat/CRISPR-associated （CRISPR/Cas） system have been successfully used in cultured human...Targeted genome modifications with the Cas9/gRNA system derived from the clustered regularly interspaced short palin- dromic repeat/CRISPR-associated （CRISPR/Cas） system have been successfully used in cultured human cells as well as in most model organisms, including zebrafish （Danio rerio）, mouse, and fruit fly （Chang et al., 2013; Cong et al., 2013; Gratz et al., 2013; Hwang et al., 2013; Jao et al., 2013; Shen et al., 2013; Wei et al., 2013）. Its application in zebrafish is particu- larly attractive due to the ease of handling this organism and the simple application of this method by direct injection of Cas9/ gRNA. However, the information about its specificity in this organism is very limited and needs further evaluation. In addition, it is conceivable that a Cas9 mRNA optimized for zebrafish codon preference could enhance its activity.展开更多
Penicillium digitatum is the most important pathogen of postharvest citrus. Gene targeting can be done in P. digitatum using homologous recombination via Agrobacterium tumefaciens mediated transformation （ATMT）, but...Penicillium digitatum is the most important pathogen of postharvest citrus. Gene targeting can be done in P. digitatum using homologous recombination via Agrobacterium tumefaciens mediated transformation （ATMT）, but the frequencies are often very low. In the present study, we replaced the Ku80 homolog （a gene of the non-homologous end-joining （NHEJ） pathway） with the hygromycin resistance cassette （hph） by ATMT. No significant change in vegetative growth, conidiation, or pathogenicity was observed in KuSO-deficient strain （△dKuSO） of P. digitatum. However, using △pdKuSO as a targeting strain, the gene-targeting frequencies for both genes PdbrlA and PdmpkA were significantly increased. These results suggest that Ku80 plays an important role in homologous inte- gration and the created △PdKuSO strain would be a good candidate for rapid gene function analysis in P. digitatum.展开更多
Functional biological research has benefited tremendously by analyses of the phenotypes of mutant organisms which can be generated through targeted mutation of genes.In Drosophila,compared with random mutagenesis meth...Functional biological research has benefited tremendously by analyses of the phenotypes of mutant organisms which can be generated through targeted mutation of genes.In Drosophila,compared with random mutagenesis methods gene targeting has gained its popularity because it can introduce any desired mutation into a gene of interest.However,applications of gene targeting have been limited because the targeting efficiency varies with different genes,and the time and labor of targeting procedure are intensive.Nevertheless,improvement of gene targeting and development of its variant technologies have received much attention of scientists.Here we review recent progress that has been made in expanding the applications of gene targeting,which include the ϕC31 integration system and zinc-finger nucleases induced gene targeting,and new strategies that generate more efficient and reliable gene targeting.展开更多
Haemophilus parasuis is one kind of constant bacteria in porcine upper respiratory tract, and it can cause multiple serositis, arthritis and other diseases under certain conditions. Due to lack of efficient genetic op...Haemophilus parasuis is one kind of constant bacteria in porcine upper respiratory tract, and it can cause multiple serositis, arthritis and other diseases under certain conditions. Due to lack of efficient genetic operating system, its pathogenic mechanism is not very clear. Ligation with DNA ligase and fusion PCR were used to construct targeting hhdA gene of Haemophilus parasuis, respectively. The fidelity, application scope, operation and conditions of the constructed fusion fragments were compared. The results showed that construction with DNA ligase was more mature technology as manifested by more stable conditions and more extensive application. The fusion PCR method had high fidelity and simple operation, and the transformation rate was 9.5 times as high as that of ligation with DNA ligase. For this reason, this method was more suitable for construction of multi-fragment targeting genes. The study lays a foundation for establishing an efficient operating system of targeting gene of Haemophilus parasuis in the future.展开更多
Edible plant derived exosome-like nanoparticles(ELNs)have been shown to have multiple nutraceutical functions.However,the diversity of plant materials makes the plant derived ELN study challenging.More efforts are sti...Edible plant derived exosome-like nanoparticles(ELNs)have been shown to have multiple nutraceutical functions.However,the diversity of plant materials makes the plant derived ELN study challenging.More efforts are still needed to explore the feasible isolation methods of edible plant derived ELNs and the possible roles of food-derived ELNs in improving human health.In this study,a size exclusion chromatography based method was compared with the traditional ultracentrifugation method to isolate blueberry derived ELNs(B-ELNs),and the miRNA profile of B-ELNs was analyzed by high-throughput sequencing.A total of 36 miRNAs were found to be enriched in B-ELNs compared with berry tissue,and their potential cross-kingdom human gene targets were further predicted.Results showed that size exclusion chromatography was effective for B-ELN isolation.The most abundant miRNAs in B-ELNs mainly belonged to the miR166 family and miR396 family.Target gene prediction indicated that B-ELNs could potentially regulate pathways related to the human digestive system,immune system and infectious diseases.展开更多
Hepatocellular carcinoma(HCC)is the third leading cause of cancer-related deaths worldwide.Major treatments include liver transplantation,resection,and chemotherapy,but the 5-year recurrence rate remains high.Late dia...Hepatocellular carcinoma(HCC)is the third leading cause of cancer-related deaths worldwide.Major treatments include liver transplantation,resection,and chemotherapy,but the 5-year recurrence rate remains high.Late diagnosis often prevents surgical intervention,contributing to poor patient survival rates.Carcinogenesis in HCC involves genetic alterations that drive the transformation of normal cells into malignant ones.Enhancer of zeste homolog 2(EZH2),a key regulator of cell cycle progression,is frequently upregulated in HCC and is associated with advanced stages and poor prognosis,making it a potential biomarker.Additionally,signal transducer and activator of transcription 3,which binds to EZH2,affects disease staging and outcomes.Targeting EZH2 presents a promising therapeutic strategy.On the other hand,abnormal lipid metabolism is a hallmark of HCC and impacts prognosis.Fatty acid binding protein 5 is highly expressed in HCC tissues and correlates with key oncogenes,suggesting its potential as a biomarker.Other genes such as guanine monophosphate synthase,cell division cycle associated 5,and epidermal growth factor receptor provide insights into the molecular mechanisms of HCC,offering potential as biomarkers and therapeutic targets.展开更多
Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the...Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community.展开更多
Objective: The mortality and morbidity rates associated with pancreatic cancer (PaCa) are extremely high. Various studies have demonstrated that pancreatic cancer will be the fourth cancer-related death by 2030, raisi...Objective: The mortality and morbidity rates associated with pancreatic cancer (PaCa) are extremely high. Various studies have demonstrated that pancreatic cancer will be the fourth cancer-related death by 2030, raising more concern for scholars to find effective methods to prevent and treat in order to improve the pancreatic cancer outcome. Using bioinformatic analysis, this study aims to pinpoint key genes that could impact PaCa patients’ prognosis and could be used as therapeutic targets. Methods: The TCGA and GEO datasets were integratively analyzed to identify prognosis-related differentially expressed genes. Next, the STRING database was used to develop PPI networks, and the MCODE and CytoNCA Cytoscape in Cytoscape were used to screen for critical genes. Through CytoNCA, three kinds of topology analysis were considered (degree, betweenness, and eigenvector). Essential genes were confirmed as potential target treatment through Go function and pathways enrichment analysis, a developed predictive risk model based on multivariate analysis, and the establishment of nomograms using the clinical information. Results: Overall, the GSE183795 and TCGA datasets associated 1311 and 2244 genes with pancreatic cancer prognosis, respectively. We identified 132 genes that were present in both datasets. The PPI network analysis using, the centrality analysis approach with the CytoNCA plug-in, showed that CDK2, PLK1, CCNB1, and TOP2A ranked in the top 5% across all three metrics. The independent analysis of a risk model revealed that the four key genes had a Hazard Ratio (HR) > 1. The monogram showed the predictive risk model and individual patient survival predictions were accurate. The results indicate that the effect of the selected vital genes was significant and that they could be used as biomarkers to predict a patient’s outcome and as possible target therapy in patients with pancreatic cancer. GO function and pathway analysis demonstrated that crucial genes might affect the P53 signaling pathway and FoxO signaling pathway, through which Meiotic nuclear division and cell cycle may have a significant function in essential genes affecting the outcome of patients who have pancreatic cancer. Conclusions: This study suggests that CDK2, CCNB1, PLK1 and TOP2A are four key genes that have a significant influence on PaCa migration and proliferation. CDK2, CCNB1, PLK1, and TOP2A can be used as potential PaCa prognostic biomarkers and therapeutic targets. However, experimental validation is necessary to confirm these predictions. Our study comes into contributions to the development of personalized target therapy for pancreatic cancer patients.展开更多
Targeted gene therapy may revolutionize disease treatment by precisely treating genetic defects.This method targets particular cells or tissues with therapeutic genes to treat a variety of genetic problems,including c...Targeted gene therapy may revolutionize disease treatment by precisely treating genetic defects.This method targets particular cells or tissues with therapeutic genes to treat a variety of genetic problems,including cancer,hereditary diseases,and viral infections.Viral,nonviral,and genome editing techniques such as CRISPR-Cas9 are used for targeted gene therapy to fix or modify disease-causing genes with minimal off-target effects.The issues of vector immunogenicity,off-target mutations,and gene delivery to target cells persist despite tremendous progress.The successful implementation of targeted gene therapy is further hindered by hereditary illness complexity and genetic background diversity.These difficulties require multidisciplinary cooperation,novel vector design,and thorough preclinical and clinical assessments.The long-term and unforeseen effects of gene editing must also be considered from an ethical viewpoint.Targeted gene therapy has considerable therapeutic promise,but more research and technological advances are needed to overcome limitations and develop safe and successful clinical treatments.展开更多
Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been th...Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were Produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An 'all-fish' gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (goGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate ho- mogeneous strain of valuable transgenic fish to fulfil human requirement in 21st century展开更多
Metabolism is a general term for a series of ordered chemical reactions in an organism used to maintain life,mainly divided into anabolic and catabolic metabolism.Nucleic acid therapy can not only precisely up-regulat...Metabolism is a general term for a series of ordered chemical reactions in an organism used to maintain life,mainly divided into anabolic and catabolic metabolism.Nucleic acid therapy can not only precisely up-regulate and down-regulate the expression of target genes but also correct mutated disease-causing genes,which demonstrates irreplaceable and outstanding advantages in the treatment of metabolismrelated diseases and has been applied to the clinical treatment of metabolism-related diseases.In this review,we introduce the structures of several major nucleic acid drugs and the mechanism of nucleic acid therapy.Subsequently,we describe the mechanisms of various biomolecular and tissue metabolisms and the etiology of metabolic disorders,classified according to metabolic substrates.We analyze the signal pathways and potential targets affecting the metabolism of each substrate and describe the nucleic acid drugs applied to these targets and their delivery technologies.This review aims to provide new ideas and targets for treating these diseases by investigating the role played by metabolism in developing diseases and providing guidance for the selection and design of nucleic acid drugs.展开更多
Alzheimer＇s disease（AD） is the most common form of dementia in the older population, however, the precise cause of the disease is unknown. The neuropathology is characterized by the presence of aggregates formed by...Alzheimer＇s disease（AD） is the most common form of dementia in the older population, however, the precise cause of the disease is unknown. The neuropathology is characterized by the presence of aggregates formed by amyloid-β（Aβ） peptide and phosphorylated tau; which is accompanied by progressive impairment of memory. Diverse signaling pathways are linked to AD, and among these the Wnt signaling pathway is becoming increasingly relevant, since it plays essential roles in the adult brain. Initially, Wnt signaling activation was proposed as a neuroprotective mechanism against Aβ toxicity. Later, it was reported that it participates in tau phosphorylation and processes of learning and memory. Interestingly, in the last years we demonstrated that Wnt signaling is fundamental in amyloid precursor protein（APP） processing and that Wnt dysfunction results in Aβ production and aggregation in vitro. Recent in vivo studies reported that loss of canonical Wnt signaling exacerbates amyloid deposition in a transgenic（Tg） mouse model of AD. Finally, we showed that inhibition of Wnt signaling in a Tg mouse previously at the appearance of AD signs, resulted in memory loss, tau phosphorylation and Aβ formation and aggregation; indicating that Wnt dysfunction accelerated the onset of AD. More importantly, Wnt signaling loss promoted cognitive impairment, tau phosphorylation and Aβ1–42 production in the hippocampus of wild-type（WT） mice, contributing to the development of an Alzheimer＇s-like neurophatology. Therefore, in this review we highlight the importance of Wnt/β-catenin signaling dysfunction in the onset of AD and propose that the loss of canonical Wnt signaling is a triggering factor of AD.展开更多
The prognosis of pancreatic carcinoma is disappointing due to the difficulty of early and accurate diagnosis,low operative resection rate, insensitivity to radiation therapy and chemotherapy. The incidence of pancreat...The prognosis of pancreatic carcinoma is disappointing due to the difficulty of early and accurate diagnosis,low operative resection rate, insensitivity to radiation therapy and chemotherapy. The incidence of pancreatic carcinoma has increased considerably in the past few years, the progress of diagnosis and treatment, however, has not been significantly improved. The overall 5-year survive rate is still as low as 5%-10%, and the improved 5 years survive rate is about 20%-40% after successive Whipple-operation. The early diagnosis is critical to the successful surgical treatment. It depends on the establishment of the new way for that.展开更多
AIM To evaluate associations between mi RNA target genes IL12B,INSR,CCND1 and IL10 polymorphisms and gastric cancer(GC)in European population.METHODS Gene polymorphisms were analyzed in 508 controls and474 GC patients...AIM To evaluate associations between mi RNA target genes IL12B,INSR,CCND1 and IL10 polymorphisms and gastric cancer(GC)in European population.METHODS Gene polymorphisms were analyzed in 508 controls and474 GC patients from 3 tertiary centers in Germany,Lithuania and Latvia.Controls were patients from the out-patient departments,who were referred for upper endoscopy because of dyspeptic symptoms and had no history of previous malignancy.Gastric cancer(GC)patients had histopathological verification of gastric adenocarcinoma.Genomic DNA was extracted using salting out method from peripheral blood mononuclear cells.IL12B T>G(rs1368439),INSR T>C(rs1051690),CCND1 A>C(rs7177)and IL10 T>C(rs3024498)SNPs were genotyped by the real-time polymerase chain reaction.Associations between gene polymorphism and GC were evaluated using multiple logistic regression analysis with adjustment for sex,age and country of birth.RESULTS We observed similar distribution of genotypes and allelic frequencies of all polymorphisms between GC patients and controls except of INSR rs1051690.The frequency of the T allele of INSR gene was significantly higher in GC patients than in controls(23.26%and 19.19%respectively,P=0.028).CT genotype was also more prevalent in patients compared to control group(38.48%and 30.12%respectively,P<0.021).Logistic regression analysis revealed that only one polymorphism(rs1051690 in INSR gene)was associated with increased risk of GC.Carriers of CT genotype had higher odds of GC when compared to CC genotype(OR=1.45,95%PI:1.08-1.95,P=0.01).Similar association was observed in a dominant model for INSR gene,where comparison of TT+CT vs CC genotypes showed an increased risk of GC(OR=1.44,95%PI:1.08-1.90,P=0.01).Other analyzed SNPs were not associated with the presence of GC.CONCLUSION INSR rs1051690 SNP is associated with increased risk of GC,while polymorphisms in IL12B,CCND1 and IL10genes are not linked with the presence of GC.展开更多
Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in tbliar infection by many...Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in tbliar infection by many plant pathogenic fungi. However, the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood. Here, we report the establishment of an Agro- bacterium tumefaciens-mediated transformation （ATMT） system and its application to investigate two autophagy genes, VdATG8 and VdATG12, by means of targeted gene replacement and complementation. Transformation of a cotton-infecting Verticillium dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant translbnnants per 1 × 10^4 conidia. V. dahliae mutants lacking either VdATG8 or VdATGI2 exhibited reduced conidiation and impaired aerial hyphae production. Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants, compared with the wild- type and gene complemented strains. Surprisingly, in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants. These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in E dahliae.展开更多
To understand the molecular characteristics of the miR477 gene family of grape(Vvi-miR477)and to predict its target genes,the Vvi-miR477 genes were identified from previous small RNA sequencing data,then phylogenetic ...To understand the molecular characteristics of the miR477 gene family of grape(Vvi-miR477)and to predict its target genes,the Vvi-miR477 genes were identified from previous small RNA sequencing data,then phylogenetic analysis and prediction of target gene were conducted.The Vvi-miR477 family consists of two precursor sequences and three mature sequences.The miR477 family members were mostly 19-22nt in length.The sequence is relatively conservative.Vvi-MIR477a and Vvi-MIR477b are located on chromosomes 1 and 2,respectively.These precursor sequences can form the typical stable stem-loop structure.Their minimum folding free energy is−39.10 kcal/mol and−50.90 kcal/mol,respectively.The MIR477 family can be divided into three groups.The prediction of target genes showed that Vvi-miR477 targets 26S proteasome,DEAD-box,GRAS family protein,Protein Phosphatase 2C,etc.The GO function of target genes was mainly enriched to six categories.The catabolic process,carboxylic ester hydrolase activity is shown to be high.This study provided a theoretical basis for further exploration of the molecular mechanism of miR477 in grape berry ripening.展开更多
The absence of effective therapies for castration-resistant prostate cancer(CRPC) establishes the need to develop novel therapeutic modality, such as targeted gene therapy, which is ideal for the treatment of CRPC. Bu...The absence of effective therapies for castration-resistant prostate cancer(CRPC) establishes the need to develop novel therapeutic modality, such as targeted gene therapy, which is ideal for the treatment of CRPC. But its application has been limited due to lack of favorable gene vector and the reduction of "bystander effect". Consequently, scientists all over the world focus their main experimental research on the following four aspects: targeted gene, vector, transfer means and comprehensive therapy. In this paper, we reviewed the latest advances of experimental research on targeted gene therapy for prostate cancer.展开更多
基金Supported by Shandong Swine Industry Technology System and Science and Technology Planning Program for Basic Research in Qingdao City(12-1-4-14-jch)
文摘Gene targeting technology is an important means to investigate gene functions, but its efficiency of gene targeting is very low, especially for somatic cell targeting. Artificially induced double-strand breaks （DSB） and triplex forming oligonucleotide （TFO） are currently developed methods to improve the targeting efficiency. This paper summarized the basic principles, design ideas and application in gene targeting efficiency improvement of these two methods, analyzed and com- pared their characteristics, and finally proposed prospects for their future development.
基金Project supported by the ScientificResearch Foundation forReturned Overseas Chinese Scholars,Education Ministry of Chinaand the Natural Science Foundation of Zhejiang Province (No.301306), China
文摘The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL 1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.
基金supported by grants from the National Basic Research Program of China (973 program, 2012CB113900)the National Natural Science Foundation of China (31071802)the Chongqing Natural Science Foundation (2011BA1002)
文摘Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors （TALEs） contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases （TALENs）, constructed using a method called ＂unit assembly＂, specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non- homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.
基金partially supported by the National Natural Science Foundation of China (No. 31110103904)the National Program on Key Basic Research Project (973 Program) of the Ministry of Science and Technology of China (Nos. 2011CBA01000 and 2012CB945101)
文摘Targeted genome modifications with the Cas9/gRNA system derived from the clustered regularly interspaced short palin- dromic repeat/CRISPR-associated （CRISPR/Cas） system have been successfully used in cultured human cells as well as in most model organisms, including zebrafish （Danio rerio）, mouse, and fruit fly （Chang et al., 2013; Cong et al., 2013; Gratz et al., 2013; Hwang et al., 2013; Jao et al., 2013; Shen et al., 2013; Wei et al., 2013）. Its application in zebrafish is particu- larly attractive due to the ease of handling this organism and the simple application of this method by direct injection of Cas9/ gRNA. However, the information about its specificity in this organism is very limited and needs further evaluation. In addition, it is conceivable that a Cas9 mRNA optimized for zebrafish codon preference could enhance its activity.
基金supported by the National Natural Science Foundation of China(Nos.31371961 and 31071649)the China Agriculture Research System(No.CARS-27)the Special Fund for Agro-scientific Research in the Public Interest(No.201203034),China
文摘Penicillium digitatum is the most important pathogen of postharvest citrus. Gene targeting can be done in P. digitatum using homologous recombination via Agrobacterium tumefaciens mediated transformation （ATMT）, but the frequencies are often very low. In the present study, we replaced the Ku80 homolog （a gene of the non-homologous end-joining （NHEJ） pathway） with the hygromycin resistance cassette （hph） by ATMT. No significant change in vegetative growth, conidiation, or pathogenicity was observed in KuSO-deficient strain （△dKuSO） of P. digitatum. However, using △pdKuSO as a targeting strain, the gene-targeting frequencies for both genes PdbrlA and PdmpkA were significantly increased. These results suggest that Ku80 plays an important role in homologous inte- gration and the created △PdKuSO strain would be a good candidate for rapid gene function analysis in P. digitatum.
文摘Functional biological research has benefited tremendously by analyses of the phenotypes of mutant organisms which can be generated through targeted mutation of genes.In Drosophila,compared with random mutagenesis methods gene targeting has gained its popularity because it can introduce any desired mutation into a gene of interest.However,applications of gene targeting have been limited because the targeting efficiency varies with different genes,and the time and labor of targeting procedure are intensive.Nevertheless,improvement of gene targeting and development of its variant technologies have received much attention of scientists.Here we review recent progress that has been made in expanding the applications of gene targeting,which include the ϕC31 integration system and zinc-finger nucleases induced gene targeting,and new strategies that generate more efficient and reliable gene targeting.
基金supported by the grants of the Major State Basic Research Development Program (2006CB504403)the Supporting Program of the"Eleventh Five-year Plan"for Sci & Tech Research of China (2006BAD06A01 and 2006BAD06A12 )+1 种基金the Foundation Project of State key Laboratory of Veterinary Etiological Biology (SKLVEB2008ZZKT009)the Open Foundation Project of Guangdong Common Lab of Veterinary Public Health (GSKJ090202)
文摘Haemophilus parasuis is one kind of constant bacteria in porcine upper respiratory tract, and it can cause multiple serositis, arthritis and other diseases under certain conditions. Due to lack of efficient genetic operating system, its pathogenic mechanism is not very clear. Ligation with DNA ligase and fusion PCR were used to construct targeting hhdA gene of Haemophilus parasuis, respectively. The fidelity, application scope, operation and conditions of the constructed fusion fragments were compared. The results showed that construction with DNA ligase was more mature technology as manifested by more stable conditions and more extensive application. The fusion PCR method had high fidelity and simple operation, and the transformation rate was 9.5 times as high as that of ligation with DNA ligase. For this reason, this method was more suitable for construction of multi-fragment targeting genes. The study lays a foundation for establishing an efficient operating system of targeting gene of Haemophilus parasuis in the future.
基金supported by the National Natural Science Foundation of China(31701561)。
文摘Edible plant derived exosome-like nanoparticles(ELNs)have been shown to have multiple nutraceutical functions.However,the diversity of plant materials makes the plant derived ELN study challenging.More efforts are still needed to explore the feasible isolation methods of edible plant derived ELNs and the possible roles of food-derived ELNs in improving human health.In this study,a size exclusion chromatography based method was compared with the traditional ultracentrifugation method to isolate blueberry derived ELNs(B-ELNs),and the miRNA profile of B-ELNs was analyzed by high-throughput sequencing.A total of 36 miRNAs were found to be enriched in B-ELNs compared with berry tissue,and their potential cross-kingdom human gene targets were further predicted.Results showed that size exclusion chromatography was effective for B-ELN isolation.The most abundant miRNAs in B-ELNs mainly belonged to the miR166 family and miR396 family.Target gene prediction indicated that B-ELNs could potentially regulate pathways related to the human digestive system,immune system and infectious diseases.
文摘Hepatocellular carcinoma(HCC)is the third leading cause of cancer-related deaths worldwide.Major treatments include liver transplantation,resection,and chemotherapy,but the 5-year recurrence rate remains high.Late diagnosis often prevents surgical intervention,contributing to poor patient survival rates.Carcinogenesis in HCC involves genetic alterations that drive the transformation of normal cells into malignant ones.Enhancer of zeste homolog 2(EZH2),a key regulator of cell cycle progression,is frequently upregulated in HCC and is associated with advanced stages and poor prognosis,making it a potential biomarker.Additionally,signal transducer and activator of transcription 3,which binds to EZH2,affects disease staging and outcomes.Targeting EZH2 presents a promising therapeutic strategy.On the other hand,abnormal lipid metabolism is a hallmark of HCC and impacts prognosis.Fatty acid binding protein 5 is highly expressed in HCC tissues and correlates with key oncogenes,suggesting its potential as a biomarker.Other genes such as guanine monophosphate synthase,cell division cycle associated 5,and epidermal growth factor receptor provide insights into the molecular mechanisms of HCC,offering potential as biomarkers and therapeutic targets.
文摘Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community.
文摘Objective: The mortality and morbidity rates associated with pancreatic cancer (PaCa) are extremely high. Various studies have demonstrated that pancreatic cancer will be the fourth cancer-related death by 2030, raising more concern for scholars to find effective methods to prevent and treat in order to improve the pancreatic cancer outcome. Using bioinformatic analysis, this study aims to pinpoint key genes that could impact PaCa patients’ prognosis and could be used as therapeutic targets. Methods: The TCGA and GEO datasets were integratively analyzed to identify prognosis-related differentially expressed genes. Next, the STRING database was used to develop PPI networks, and the MCODE and CytoNCA Cytoscape in Cytoscape were used to screen for critical genes. Through CytoNCA, three kinds of topology analysis were considered (degree, betweenness, and eigenvector). Essential genes were confirmed as potential target treatment through Go function and pathways enrichment analysis, a developed predictive risk model based on multivariate analysis, and the establishment of nomograms using the clinical information. Results: Overall, the GSE183795 and TCGA datasets associated 1311 and 2244 genes with pancreatic cancer prognosis, respectively. We identified 132 genes that were present in both datasets. The PPI network analysis using, the centrality analysis approach with the CytoNCA plug-in, showed that CDK2, PLK1, CCNB1, and TOP2A ranked in the top 5% across all three metrics. The independent analysis of a risk model revealed that the four key genes had a Hazard Ratio (HR) > 1. The monogram showed the predictive risk model and individual patient survival predictions were accurate. The results indicate that the effect of the selected vital genes was significant and that they could be used as biomarkers to predict a patient’s outcome and as possible target therapy in patients with pancreatic cancer. GO function and pathway analysis demonstrated that crucial genes might affect the P53 signaling pathway and FoxO signaling pathway, through which Meiotic nuclear division and cell cycle may have a significant function in essential genes affecting the outcome of patients who have pancreatic cancer. Conclusions: This study suggests that CDK2, CCNB1, PLK1 and TOP2A are four key genes that have a significant influence on PaCa migration and proliferation. CDK2, CCNB1, PLK1, and TOP2A can be used as potential PaCa prognostic biomarkers and therapeutic targets. However, experimental validation is necessary to confirm these predictions. Our study comes into contributions to the development of personalized target therapy for pancreatic cancer patients.
文摘Targeted gene therapy may revolutionize disease treatment by precisely treating genetic defects.This method targets particular cells or tissues with therapeutic genes to treat a variety of genetic problems,including cancer,hereditary diseases,and viral infections.Viral,nonviral,and genome editing techniques such as CRISPR-Cas9 are used for targeted gene therapy to fix or modify disease-causing genes with minimal off-target effects.The issues of vector immunogenicity,off-target mutations,and gene delivery to target cells persist despite tremendous progress.The successful implementation of targeted gene therapy is further hindered by hereditary illness complexity and genetic background diversity.These difficulties require multidisciplinary cooperation,novel vector design,and thorough preclinical and clinical assessments.The long-term and unforeseen effects of gene editing must also be considered from an ethical viewpoint.Targeted gene therapy has considerable therapeutic promise,but more research and technological advances are needed to overcome limitations and develop safe and successful clinical treatments.
文摘Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were Produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An 'all-fish' gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (goGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate ho- mogeneous strain of valuable transgenic fish to fulfil human requirement in 21st century
基金financially supported by the National Natural Science Foundation of China(Nos.32225029,22205240,52073287,22075289,82071552 and 22376006)National Key R&D Program of China(No.2023YFC2605003)。
文摘Metabolism is a general term for a series of ordered chemical reactions in an organism used to maintain life,mainly divided into anabolic and catabolic metabolism.Nucleic acid therapy can not only precisely up-regulate and down-regulate the expression of target genes but also correct mutated disease-causing genes,which demonstrates irreplaceable and outstanding advantages in the treatment of metabolismrelated diseases and has been applied to the clinical treatment of metabolism-related diseases.In this review,we introduce the structures of several major nucleic acid drugs and the mechanism of nucleic acid therapy.Subsequently,we describe the mechanisms of various biomolecular and tissue metabolisms and the etiology of metabolic disorders,classified according to metabolic substrates.We analyze the signal pathways and potential targets affecting the metabolism of each substrate and describe the nucleic acid drugs applied to these targets and their delivery technologies.This review aims to provide new ideas and targets for treating these diseases by investigating the role played by metabolism in developing diseases and providing guidance for the selection and design of nucleic acid drugs.
基金supported by grants PFB (Basal Financing Program) 12/2007 from the Basal Centre for Excellence in Science and Technology and FONDECYT,No.1120156(to NCI)a pre-doctoral fellowship from the National Commission of Science and Technology of Chile(CONICYT)(to CTR)
文摘Alzheimer＇s disease（AD） is the most common form of dementia in the older population, however, the precise cause of the disease is unknown. The neuropathology is characterized by the presence of aggregates formed by amyloid-β（Aβ） peptide and phosphorylated tau; which is accompanied by progressive impairment of memory. Diverse signaling pathways are linked to AD, and among these the Wnt signaling pathway is becoming increasingly relevant, since it plays essential roles in the adult brain. Initially, Wnt signaling activation was proposed as a neuroprotective mechanism against Aβ toxicity. Later, it was reported that it participates in tau phosphorylation and processes of learning and memory. Interestingly, in the last years we demonstrated that Wnt signaling is fundamental in amyloid precursor protein（APP） processing and that Wnt dysfunction results in Aβ production and aggregation in vitro. Recent in vivo studies reported that loss of canonical Wnt signaling exacerbates amyloid deposition in a transgenic（Tg） mouse model of AD. Finally, we showed that inhibition of Wnt signaling in a Tg mouse previously at the appearance of AD signs, resulted in memory loss, tau phosphorylation and Aβ formation and aggregation; indicating that Wnt dysfunction accelerated the onset of AD. More importantly, Wnt signaling loss promoted cognitive impairment, tau phosphorylation and Aβ1–42 production in the hippocampus of wild-type（WT） mice, contributing to the development of an Alzheimer＇s-like neurophatology. Therefore, in this review we highlight the importance of Wnt/β-catenin signaling dysfunction in the onset of AD and propose that the loss of canonical Wnt signaling is a triggering factor of AD.
文摘The prognosis of pancreatic carcinoma is disappointing due to the difficulty of early and accurate diagnosis,low operative resection rate, insensitivity to radiation therapy and chemotherapy. The incidence of pancreatic carcinoma has increased considerably in the past few years, the progress of diagnosis and treatment, however, has not been significantly improved. The overall 5-year survive rate is still as low as 5%-10%, and the improved 5 years survive rate is about 20%-40% after successive Whipple-operation. The early diagnosis is critical to the successful surgical treatment. It depends on the establishment of the new way for that.
基金Supported by Lithuanian Research Council Grant,No.MIP-14418
文摘AIM To evaluate associations between mi RNA target genes IL12B,INSR,CCND1 and IL10 polymorphisms and gastric cancer(GC)in European population.METHODS Gene polymorphisms were analyzed in 508 controls and474 GC patients from 3 tertiary centers in Germany,Lithuania and Latvia.Controls were patients from the out-patient departments,who were referred for upper endoscopy because of dyspeptic symptoms and had no history of previous malignancy.Gastric cancer(GC)patients had histopathological verification of gastric adenocarcinoma.Genomic DNA was extracted using salting out method from peripheral blood mononuclear cells.IL12B T>G(rs1368439),INSR T>C(rs1051690),CCND1 A>C(rs7177)and IL10 T>C(rs3024498)SNPs were genotyped by the real-time polymerase chain reaction.Associations between gene polymorphism and GC were evaluated using multiple logistic regression analysis with adjustment for sex,age and country of birth.RESULTS We observed similar distribution of genotypes and allelic frequencies of all polymorphisms between GC patients and controls except of INSR rs1051690.The frequency of the T allele of INSR gene was significantly higher in GC patients than in controls(23.26%and 19.19%respectively,P=0.028).CT genotype was also more prevalent in patients compared to control group(38.48%and 30.12%respectively,P<0.021).Logistic regression analysis revealed that only one polymorphism(rs1051690 in INSR gene)was associated with increased risk of GC.Carriers of CT genotype had higher odds of GC when compared to CC genotype(OR=1.45,95%PI:1.08-1.95,P=0.01).Similar association was observed in a dominant model for INSR gene,where comparison of TT+CT vs CC genotypes showed an increased risk of GC(OR=1.44,95%PI:1.08-1.90,P=0.01).Other analyzed SNPs were not associated with the presence of GC.CONCLUSION INSR rs1051690 SNP is associated with increased risk of GC,while polymorphisms in IL12B,CCND1 and IL10genes are not linked with the presence of GC.
基金supported by the grants from the State Key Basic Research and Development Plan of China(No. 2011CB109300)the National Natural Science Foundation of China(No.31171590)+2 种基金Jiangsu Province Natural Science Foundation(Nos.BK2010065 and BE2012329)the Specialized Research Fund for the Doctoral Program of Higher Education of China(No.20090097110010)the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘Autophagy is a widely conserved intracellular process for degradation and recycling of proteins, organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in tbliar infection by many plant pathogenic fungi. However, the role of autophagy in soil-borne fungal physiology and infection biology is poorly understood. Here, we report the establishment of an Agro- bacterium tumefaciens-mediated transformation （ATMT） system and its application to investigate two autophagy genes, VdATG8 and VdATG12, by means of targeted gene replacement and complementation. Transformation of a cotton-infecting Verticillium dahliae strain Vd8 with a novel binary vector pCOM led to the production of 384 geneticin-resistant translbnnants per 1 × 10^4 conidia. V. dahliae mutants lacking either VdATG8 or VdATGI2 exhibited reduced conidiation and impaired aerial hyphae production. Disease development on Arabidopsis plants was slightly delayed when inoculated with VdATG8 or VdATG12 gene deletion mutants, compared with the wild- type and gene complemented strains. Surprisingly, in vitro inoculation with unimpaired roots revealed that the abilities of root invasion were not affected in gene deletion mutants. These results indicate that autophagy is necessary for aerial hyphae development and plant colonization but not for root infection in E dahliae.
基金This work was financially supported by National Key Research and Development Program of China(2018YFD1000105)Natural Science Foundation of China(NSFC:U1904113)Program for Innovative Research Team(in Science and Technology)in University of Henan Province(21IRTSTHN021),China.
文摘To understand the molecular characteristics of the miR477 gene family of grape(Vvi-miR477)and to predict its target genes,the Vvi-miR477 genes were identified from previous small RNA sequencing data,then phylogenetic analysis and prediction of target gene were conducted.The Vvi-miR477 family consists of two precursor sequences and three mature sequences.The miR477 family members were mostly 19-22nt in length.The sequence is relatively conservative.Vvi-MIR477a and Vvi-MIR477b are located on chromosomes 1 and 2,respectively.These precursor sequences can form the typical stable stem-loop structure.Their minimum folding free energy is−39.10 kcal/mol and−50.90 kcal/mol,respectively.The MIR477 family can be divided into three groups.The prediction of target genes showed that Vvi-miR477 targets 26S proteasome,DEAD-box,GRAS family protein,Protein Phosphatase 2C,etc.The GO function of target genes was mainly enriched to six categories.The catabolic process,carboxylic ester hydrolase activity is shown to be high.This study provided a theoretical basis for further exploration of the molecular mechanism of miR477 in grape berry ripening.
文摘The absence of effective therapies for castration-resistant prostate cancer(CRPC) establishes the need to develop novel therapeutic modality, such as targeted gene therapy, which is ideal for the treatment of CRPC. But its application has been limited due to lack of favorable gene vector and the reduction of "bystander effect". Consequently, scientists all over the world focus their main experimental research on the following four aspects: targeted gene, vector, transfer means and comprehensive therapy. In this paper, we reviewed the latest advances of experimental research on targeted gene therapy for prostate cancer.
