Transgenic cotton carrying the CrylAc gene has revolutionized insect pest control since its adoption,although the development of resistance in insect pests has reduced its efficacy.After 10 years of cultivating Bacill...Transgenic cotton carrying the CrylAc gene has revolutionized insect pest control since its adoption,although the development of resistance in insect pests has reduced its efficacy.After 10 years of cultivating Bacillus thuringiensis(Bt)cotton with a single Cry1 Ac gene,growers are on the verge of adopting Bt cotton that carries the double gene(Cry1 Ac+Cry2 A)due to its better effectiveness against insect pests.Thus,the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene.The expression levels of the Cry1 Ac and Cry2 A genes were evaluated in the leaves of 10 genotypes(2 parents and 8 Fhybrids)at 30 days after sowing(DAS),while samples of leaves,bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS.The Fhybrids were developed through reciprocal crosses between two Bt(CKC-1,CKC-2)and two non-Bt(MNH-786,FH-942)parents.The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay(ELISA).The results showed that the MNH786 xCKC-1 hybrid had the highest concentrations of Cry1 Ac gene at30 DAS(3.08μg g^(-1))and 110 DAS(1.01μg g^(-1))in leaves.In contrast,the CKC-2 xMNH-786 hybrid showed the lowest concentrations of Cry1 Ac gene at 30 DAS(2.30μg g^(-1))and 110 DAS(0.86μg g^(-1)).The Fhybrid FH-942×CKC-2 showed the highest concentrations of Cry2 A gene at 30 DAS(8.39μg g^(-1))and 110 DAS(7.74μg g^(-1))in leaves,while the CKC-1 xMNH-786 hybrid expressed the lowest concentrations of Cry2 A gene at 30 DAS(7.10μg g^(-1))and 110 DAS(8.31μg g^(-1)).A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS,whereas the lowest concentrations were observed at 110 DAS for both genes in leaves.When the expression pattern was compared between various plant parts in genotype CKC-2,it was found that leaves had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))at 70 DAS,followed by bolls(Cry1 Ac(1.66μg g^(-1))and Cry2 A(8.15μg g^(-1)))and flowers(Cry1 Ac(1.07μg g^(-1))and Cry2 A(7.99μg g^(-1))).The genotype CKC-2 had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))in the upper canopy but less accumulation(2.66μg g^(-1)of Cry1 Ac,8.09μg g^(-1)of Cry2 A)in the lower canopy at 70 DAS.Similarly,at 110 DAS,the expression levels of Cry1 Ac and Cry2 A in upper and lower canopy leaves were 1.52 and 7.92μg 9,and 0.99 and 7.54μg 9,respectively.Hence,the current study demonstrates that different genotypes showed variable expression for both of the Cry1 Ac and Cry2 A genes during plant growth due to different genetic backgrounds.The Cry2 A gene had three-fold higher expression than Cry1 Ac with significant differences in expression in different plant parts.The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1 Ac and Cry2 A for sustainable cotton production worldwide.展开更多
A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, desig...A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.展开更多
Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard met...Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard methods of shRNA transgenic (Tg) mice production have not been established. Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. Then high efficiency of Sry gene silencing in Tg mice should induce XY male-to-female sex reversal. An shRNA Tg mouse targeting Sry gene was attempted to be generated by pronuclear microinjection. A low rate (Tg pups/all pups born after microinjection = 2/154 to 7/178) of Tg pups was observed. These Tg mice showed no XY male-to-female sex reversal. The results suggest that exogenous expression of small RNA might exert a negative effect on embryonic development and another approach should be needed for RNAi transgenesis in mice.展开更多
Over the last few decades, dengue fever epidemics have increased in frequency and intensity worldwide, making it a major global concern for public health. Its prevention, which is essentially vector-based control, is ...Over the last few decades, dengue fever epidemics have increased in frequency and intensity worldwide, making it a major global concern for public health. Its prevention, which is essentially vector-based control, is already being compromised by reports of resistance of the main vector Aedes aegypti to insecticides. To tackle the rapid increase in insecticide resistance and outbreaks, the biological vector control is a promising approach. One of the strategies of this approach is the use of entomopathogenic fungi because of their great efficacy and their eco-friendly aspects. However, some aspects of their use, such as the low efficiency, the high cost of production and the sensitivity to various adverse conditions, need to be addressed for their successful large-scale application. Therefore, innovative technologies based on strains of transgenic fungi with improved biocontrol potentials by genetic engineering are actively pursued. Although these modified mycoinsecticides are acclaimed for their better effectiveness against target insects, the main concern remains their potential adverse effects on the environment and human health. The present review is dedicated to giving an update on recent developments in transgenic entomopathogenic fungi (TEF) for Aedes mosquito control. Future perspectives are also proposed to address the safety concerns related to the release of transgenic entomopathogenic fungi into the environment.展开更多
Chemicals that modify pest behavior are developed to reduce crop damage by altering pest behavior, using specific genes within the olfactory system as molecular targets. The identification of these molecular targets i...Chemicals that modify pest behavior are developed to reduce crop damage by altering pest behavior, using specific genes within the olfactory system as molecular targets. The identification of these molecular targets in Bactrocera dorsalis, also known as the functional study of key olfactory genes, relies on CRISPR/Cas9-mediated gene knockout techniques. However, these techniques face limitations when applied to lethal genes. Transgenic technology offers a solution since it enables precise manipulation of gene expression in specific tissues or during certain developmental stages. Consequently, this study developed a piggyBac-mediated transgenic system in B. dorsalis to investigate reporter gene expression in olfactory organs, and assessed the olfactory behavior andantennal electrophysiological responses in transgenic lines. The goal was to assess the potential of this approach for future research on olfactory gene function. A universally expressed housekeeping gene from the BdorActin family was identified using the developmental transcriptome dataset. Its candidate promoter region(BdorActinA3a-1^(P–2k)) was then cloned into the piggyBac plasmid. We subsequently established two stable transgenic lines with specific TTAA insertion sites on chromosomes 4 and 5, consistent with the characteristics of piggyBac transposition. The transgenic strains exhibited essentially normal survival, with hatchability and adult lifespan unaffected, althoughthere were slight reductions in the emergence rate and oviposition capacity. The fluorescent reporter has been successfully expressed in olfactory-related organs, such as the antennae, proboscis, maxillary palp, legs, external genitalia, and brain. The antennal electrophysiological responses to representative chemicals in the transgenic lines were consistent with those of the wild type. However, some olfactory-related behaviors, such as pheromone response and mating, were significantly affected in the transgenic lines. These findings suggest that our system could potentially be applied in future olfactory research, such as driving the expression of exogenous elements that are effective in olfactory organs. However, caution is advised regarding its impact when applied to some olfactoryrelated behavioral phenotypes.展开更多
In addition to the negative consequences of climate change,sucking pest complexes severely limited cotton yields in the recent past.Although the damage caused by bollworms was much reduced by utilizing Bt cotton,the e...In addition to the negative consequences of climate change,sucking pest complexes severely limited cotton yields in the recent past.Although the damage caused by bollworms was much reduced by utilizing Bt cotton,the emergence of sucking pests(such as aphids,thrips,and whiteflies)poses a serious threat to cotton production,as they reduce lint yield by 40%–60%finally.Additionally,these pests also caused yield losses by spreading viral diseases.Promoting innovative and thorough control methods is necessary to counter the threat posed by these sucking pests.Such initiatives necessitate a multifaceted strategy that combines next-generation breeding technology and pest management techniques to produce novel cotton cultivars that are resistant to sucking pests.The discovery of novel genes and regulatory factors linked to cotton’s resistance to sucking pests will be possible by the combination of next-generation breeding technologies and omics approaches and employing those tools on special resistant donors.Continuous research aimed at understanding the genetic basis of insect resistance and improving integrated pest management(IPM)techniques is crucial to the sustainability and resilience of cotton cropping systems.To this end,a sustainable and viable strategy to protect cotton fields from sucking pests is outlined.展开更多
Background Transgenic research in crops involves using genetic engineering techniques to introduce specific genes of interest from other organisms,or even entirely new genes into plant genomes to create crops with des...Background Transgenic research in crops involves using genetic engineering techniques to introduce specific genes of interest from other organisms,or even entirely new genes into plant genomes to create crops with desirable traits that wouldn’t be possible through conventional breeding methods.Transgenic crops have been developed for various traits globally.Whitefly,Bemisia tabaci(Gennadius)is one of the major sucking pests of cotton that cause significant damage to the cotton production.To combat whitefly infestations,researchers have developed four transgenic cotton lines expressing the fern protein.And those transgenic lines need to be evaluated for their performance against the target pest—whitefly.The evaluation was designed as controlled trials in polyhouse or muslin cloth cages under open-choice and no-choice conditions by comparing four transgenic cotton lines(A,B,C,and D)with three control groups,including untransformed cotton plants with a same genetic background of the transgenic line,conventionally bred whitefly-resistant cotton,and whitefly-susceptible cotton.In order to study the generational effect,the evaluation also involved studies on whitefly development in laboratory,muslin cloth cage,and polyhouse conditions.Results Both open-choice and no-choice experiments had shown that all the four transgenic cotton lines(A,B,C,and D)expressing the fern protein reduced adult whitefly numbers significantly compared with the control lines,except for the no-choice conditions in 2021,where the transgenic line C was non-significant different from the resistant control line.Notably,the nymphal population on the resistant control line was relatively low and nonsignificant different from the transgenic line C in 2021;and the transgenic lines A and C in 2022 under open-choice conditions.Under no-choice condition,the nymphal counts in the resistant control line was non-significant different from transgenic lines C and D in 2021;and transgenic line D in 2022.All transgenic lines showed significant decrease in egg hatching in 2021 and nymphal development in 2022,except for the transgenic line C which had no significant different in the nymphal development comparing with non-transgenic control lines in 2022.Adult emergence rates in both years of evaluation showed significant decrease in transgenic lines A and B comparing with the control lines.Additionally,the results showed a significant reduction in cotton leaf curl disease and sooty mold development in all the four transgenic lines compared with susceptible control under open-choice conditions,indicating potential benefits of transgenic lines beyond direct effect on whitefly control.Furthermore,the research explored the generational effects of the fern protein on whitefly which revealed the lowest fecundity in the transgenic line C across F0,F1 and F3 generations,lower egg hatching in F1 and F2 generations in transgenic lines A and B,shorter nymphal duration in F1 and F2 generations in transgenic line B,and the least total adult emergence in the transgenic line C in F0 and F3 generations.Conclusions These findings suggest that the transgenic cotton lines expressing fern protein disrupts whitefly populations and the life cycle to a certain extent.However,results are not consistent over generations and years of study,indicating these transgenic lines were not superior over control lines and need to be improved in future breeding.展开更多
Wheat(Triticum aestivum L.)is a highly valued cereal crop produced and consumed globally,particularly in arid or semi-arid regions(Zhou et al.,2020;Mao et al.,2023).However,its production is increasingly threatened by...Wheat(Triticum aestivum L.)is a highly valued cereal crop produced and consumed globally,particularly in arid or semi-arid regions(Zhou et al.,2020;Mao et al.,2023).However,its production is increasingly threatened by the rising incidence of drought events associated with climate change.Arid regions are especially susceptible to these droughts,which are intensifying in both severity and frequency(Eckardt et al.,2023;Mao et al.,2023;Yang and Qin,2023).As of a 2022 report,more than 92%of wheat-producing regions are estimated to experience one or more drought/heatwave events in each growing season.Furthermore,the duration and frequency of these combined stress events have increased by approximately 28%over the past four decades(He et al.,2022).To address this challenge,wheat breeding programs have allocated substantial and research efforts to developing elite,stress tolerant lines.This initiative is large part by rapid innovation in transgenic and genome editing strategies(Hu and Xiong,2014;Gao et al.,2021.展开更多
Background : Traditional DNA microinjection methods used in mammals are difficult to apply to avian species due to their unique reproductive characteristics. Genetic manipulation in chickens, particularly involving im...Background : Traditional DNA microinjection methods used in mammals are difficult to apply to avian species due to their unique reproductive characteristics. Genetic manipulation in chickens, particularly involving immature follicles within living ovaries, has not been extensively explored. This study seeks to establish an efficient method for generating transgenic chickens through ovarian injection, potentially bypassing the challenges associated with primordial germ cell (PGC) manipulation and fertilized egg microinjection. Methods : Hens were anesthetized and underwent a surgical procedure to access the ovary for DNA injection into immature follicles. The study used liposomes to deliver GFP- expressing plasmids at various dosages. After injection, hens recovered, and their eggs were fertilized through artificial insemination. Results : Transgenic chickens were successfully generated in one generation without needing G0 founders. The injection of 20 μg plasmid yielded the highest transgenic efficiency at 12.1%. GFP- positive embryos were confirmed through microscopy, and successful transgene expression was validated at the tissue level using immunostaining. TERT and GFP elements introduced in the G1 generation resulted in 4.1% positive transgene rates, as confirmed by PCR and Southern blotting. Conclusion : This ovarian injection method offers a promising alternative for avian genetic manipulation, bypassing complex PGC procedures and enabling direct generation of G1 transgenic chickens. This technique simplifies the transgenic process for chickens and has the potential to be adapted for other avian species, especially those without established PGCs culture systems.展开更多
Aluminium(Al)toxicity is one of the key factors limiting crop output in acidic soils,but until now little has been known about how Al is regulated transcriptionally in plants.This study identified Arabidopsis NAC tran...Aluminium(Al)toxicity is one of the key factors limiting crop output in acidic soils,but until now little has been known about how Al is regulated transcriptionally in plants.This study identified Arabidopsis NAC transcription factor ANAC050 in the regulation of Al tolerance.ANAC050 was located in the nucleus and displayed constitutive expression in the silique,flower,leaf,stem,and root,despite the fact that Al stress decreased its expression and protein accumulation.When compared with the Columbia ecotype wild-type,anac050 mutants that lacked function of ANAC050 exhibited Al sensitivity phenotype,while transgenic lines that overexpressed ANAC050 showed an Al-resistant phenotype,indicating the favorable influence of ANAC050 on preserving Al tolerance in plants.Further analysis indicated that anac050 mutants accumulated more Al in roots,implying that ANAC050 may confer a potential operation of an Al exclusion mechanism.Interestingly,anac050 mutants had down-regulated the expression of the genes encoding MULTIDRUG AND TOXIC COMPOUND EXTRUSION(MATE)and AL-ACTIVATED MALATE TRANSPORTER(ALMT1),which were involved in the secretion of citrate and malate,even though there was no evidence of a direct interaction between them,suggesting ANAC050 may mediate the secretion of citrate and malate indirectly.Together with the decreased hemicellulose content,lower Al content was also discovered in root cell walls and hemicelluloses of anac050 mutants,pointing to a potential interaction between ANAC017 and XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE(XTH).Although there was no evidence of a direct interaction between ANAC050 and XTH31,it is worth mentioning that the expression of XTH31,which is essential for xyloglucan modification,was down-regulated in anac050 mutants irrespective of the amount of Al given.In conclusion,our findings showed that ANAC050 contributed to Al resistance by indirect control of the release of organic acids and the accumulation of cell wall hemicelluloses.展开更多
Traditional Chinese medicine(TCM)can help prevent or treat diseases;however,there are few studies on the active substances of TCM.For example,Lycium barbarum L.has been proven to be effective in treating osteoporosis ...Traditional Chinese medicine(TCM)can help prevent or treat diseases;however,there are few studies on the active substances of TCM.For example,Lycium barbarum L.has been proven to be effective in treating osteoporosis for thousands of years,but its active substance remains to be unknown.Prompted by the efforts to modernize TCM,the present study focused on the novel active substance of Lycium barbarum L.to reinforce kidney essence to produce bone marrow.Illumina deep sequencing analysis and stemloop polymerase chain reaction(PCR)assay revealed that miR162a,a Lycium barbarum L.-derived microRNA,can pass through the gastrointestinal tract to target the bone marrow in mice.Immunofluorescence staining showed that miR162a was absorbed through systemic RNA interference defective transmembrane family member 1(SIDT1)in the stomach.Bioinformatics prediction and luciferase reporter assay identified that miR162a targeted nuclear receptor corepressor(NcoR).Alizarin red staining and micro-computed tomography(microCT)confirmed that miR162a promoted osteogenic differentiation in bone marrow mesenchymal stem cells,zebrafish,and a mouse model of osteoporosis.In addition,transgenic Nicotiana benthamiana(N.benthamiana)leaves overexpressing miR162a were developed by agrobacterium infiltration method.microCT and tartrate-resistant acid phosphatase staining confirmed that transgenic N.benthamiana leaves effectively protected against osteoporosis in mice.Our study mechanistically explains how Lycium barbarum L.improves osteoporosis and supports that Lycium barbarum L.reinforces kidney essence,thereby strengthening the bone.miR162a expressed by transgenic plants may represent a novel and safe treatment for human osteoporosis.展开更多
Cilia are indispensable for organ development and function,and their dysfunction causes a range of syndromic diseases known as ciliopathies,including obesity,cystic kidney disease,situs inversus,and male infertility(R...Cilia are indispensable for organ development and function,and their dysfunction causes a range of syndromic diseases known as ciliopathies,including obesity,cystic kidney disease,situs inversus,and male infertility(Reiter and Leroux,2017;Wallmeier et al.,2020).To date,over 180 ciliopathy-associated genes have been identified(Reiter and Leroux,2017),yet the underlying mechanisms remain poorly understood.展开更多
Parkinson’s disease is characterized by synucleinopathy-associated neurodegeneration.Previous studies have shown that glucagon-like peptide-1(GLP-1)has beneficial effects in a mouse model of Parkinson’s disease indu...Parkinson’s disease is characterized by synucleinopathy-associated neurodegeneration.Previous studies have shown that glucagon-like peptide-1(GLP-1)has beneficial effects in a mouse model of Parkinson’s disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.However,the effect of GLP-1 on intrinsic synuclein malfunction remains unclear.In this study,we investigated the effect of Lactococcus lactis MG1363-pMG36e-GLP-1 on parkinsonism in SncaA53T transgenic mice and explored the underlying mechanisms.Our data showed that Lactococcus lactis MG1363-pMG36e-GLP-1 inhibited dopaminergic neuronal death,reduced pathological aggregation ofα-synuclein,and decreased movement disorders in SncaA53T transgenic mice.Furthermore,Lactococcus lactis MG1363-pMG36e-GLP-1 downregulated lipopolysaccharide-related inflammation,reduced cerebral activation of microglia and astrocytes,and promoted cell survival via the GLP-1 receptor/PI3K/Akt pathway in the substantia nigra.Additionally,Lactococcus lactis MG1363-pMG36e-GLP-1 decreased serum levels of pro-inflammatory molecules including lipopolysaccharide,lipopolysaccharide binding protein,interleukin-1β,and interleukin-6.Gut histopathology and western blotting further revealed that Lactococcus lactis MG1363-pMG36e-GLP-1 increased the expression of gut integrity-related proteins and reduced lipopolysaccharide-related inflammation by reversing gut dysbiosis in SncaA53T transgenic mice.Our findings showed that the beneficial effect of Lactococcus lactis MG1363-pMG36e-GLP-1 on parkinsonism traits in SncaA53T transgenic mice is mediated by microglial polarization and the reversal of dysbiosis.Collectively,our findings suggest that Lactococcus lactis MG1363-pMG36e-GLP-1 is a promising therapeutic agent for the treatment of Parkinson’s disease.展开更多
It has been more than 20 years since the first batch of transgenic fish was produced. Five stable germ-line transmitted growth hormone (GH) transgenic fish lines have been generated. This paper reviews the mechanisms ...It has been more than 20 years since the first batch of transgenic fish was produced. Five stable germ-line transmitted growth hormone (GH) transgenic fish lines have been generated. This paper reviews the mechanisms of integration and gene targeting of the transgene, as well as the viability, reproduction and transgenic approaches for the reproductive containment of GH-transgenic fish. Further, we propose that it should be necessary to do the following studies, in particularly, of the breeding of transgenic fish: to assess the fitness of transgenic fish in an aqueous environment with a large space and a complex structure; and to develop a controllable on-off strategy of reproduction in transgenic fish.展开更多
To gain information on the integration pattern of pMThGH-transgene, 50 transgenes were recovered from F4 generation of pMThGH transgenic common carp (Cyprinus carpio L.) and 33 recovered genes were analyzed. The restr...To gain information on the integration pattern of pMThGH-transgene, 50 transgenes were recovered from F4 generation of pMThGH transgenic common carp (Cyprinus carpio L.) and 33 recovered genes were analyzed. The restriction maps of these recovered genes were constructed by digestion with five kinds of enzymes. These transgenes can be classified into 4 types according to their restriction maps. Only one type of transgenes maintains its original molecular form, whereas the other three types are very different from the original one and vary each other on both molecular weight and restriction maps. This implies that the sequences of most transgenes have been deleted and/or rearranged during integration and inheritance. The results of PCR amplification and Southern blot hybridization indicate that MThGH in Type I transgene keeps intact but most of its sequence has been deleted in other three types. All these results suggest that transgenes in F4 generation of transgenic carp are highly polymorphic. Two DNA展开更多
Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes.However,their activity markedly diminishes with payloads exceeding 11 kb.Expanding the p...Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes.However,their activity markedly diminishes with payloads exceeding 11 kb.Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs,improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics,metabolic engineering,and transgenic animal production.In this study,we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer(QBI SP163,ST)and enhanced the nuclear targeting ability using the nuclear localization protein H2B(SHT).The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures(H1299),comparable to the well-established super PiggyBac system.Furthermore,mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads(8 kb,14 kb,and 24 kb)into zebrafish(Danio rerio).This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.展开更多
Huntington'sdisease(HD)isahereditary neurodegenerative disorder for which there is currently no effectivetreatmentavailable.Consequently,the development of appropriate disease models is critical to thoroughly inve...Huntington'sdisease(HD)isahereditary neurodegenerative disorder for which there is currently no effectivetreatmentavailable.Consequently,the development of appropriate disease models is critical to thoroughly investigate disease progression.The genetic basis of HD involves the abnormal expansion of CAG repeats in the huntingtin(HTT)gene,leading to the expansion of a polyglutamine repeat in the HTT protein.Mutant HTT carrying the expanded polyglutamine repeat undergoes misfolding and forms aggregates in the brain,which precipitate selective neuronal loss in specific brain regions.Animal models play an important role in elucidating the pathogenesis of neurodegenerative disorders such as HD and in identifying potential therapeutic targets.Due to the marked species differences between rodents and larger animals,substantial efforts have been directed toward establishing large animal models for HD research.These models are pivotal for advancing the discovery of novel therapeutic targets,enhancing effective drug delivery methods,and improving treatment outcomes.We have explored the advantages of utilizing large animal models,particularly pigs,in previous reviews.Since then,however,significant progress has been made in developing more sophisticated animal models that faithfully replicate the typical pathology of HD.In the current review,we provide a comprehensive overview of large animal models of HD,incorporating recent findings regarding the establishment of HD knock-in(KI)pigs and their genetic therapy.We also explore the utilization of large animal models in HD research,with a focus on sheep,non-human primates(NHPs),and pigs.Our objective is to provide valuable insights into the application of these large animal models for the investigation and treatment of neurodegenerative disorders.展开更多
Selenocysteine methyltransferase(SMT)is a key enzyme involved in the Se metabolism pathway,and it is responsible for the catalysis of Se-methylselenocysteine(SeMSC)compound formation.Previous studies showed that selen...Selenocysteine methyltransferase(SMT)is a key enzyme involved in the Se metabolism pathway,and it is responsible for the catalysis of Se-methylselenocysteine(SeMSC)compound formation.Previous studies showed that selenium treatment activated SMT expression and promoted the accumulation of glucosinolates(GSLs)and sulforaphane,but the roles and functional mechanisms of SMT in mediating GSLs and sulforaphane synthesis remain unclear.In this study,we identified the BoSMT gene in broccoli and uncovered its roles in mediating GSLs biosynthesis.Transgenic assays revealed that BoSMT is involved in SeMSC biosynthesis in broccoli.More importantly,the contents of GSLs and sulforaphane were significantly increased in the BoSMT-overexpressing broccoli lines but decreased in the knockdown lines,suggesting that BoSMT played a positive role in regulating GSLs and sulforaphane synthesis.Further evidence indicated that BoSMT-mediated overaccumulation of GSLs and sulforaphane might be due to the increase in the endogenous SeMSC content.Compared with the mock(water)treatment,selenite-induced significantly increases of the SeMSC content in the BoSMT-knockdown plants partially compensated the phenotype of GSLs and sulforaphane loss.Compared with the mock treatment,exogenous SeMSC treatment significantly increased the contents of GSL and sulforaphane and activated GSL synthesis-related gene expression,suggesting that SeMSC acted as a positive regulator for GSL and sulforaphane production.Our findings provided novel insights into selenium-mediated GSLs and sulforaphane accumulation.The genetic manipulation of BoSMT might be a useful strategy for improving the dietary nutritional values of broccoli.展开更多
Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation ...Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.展开更多
基金Higher Education Commission,Pakistan for providing funds。
文摘Transgenic cotton carrying the CrylAc gene has revolutionized insect pest control since its adoption,although the development of resistance in insect pests has reduced its efficacy.After 10 years of cultivating Bacillus thuringiensis(Bt)cotton with a single Cry1 Ac gene,growers are on the verge of adopting Bt cotton that carries the double gene(Cry1 Ac+Cry2 A)due to its better effectiveness against insect pests.Thus,the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene.The expression levels of the Cry1 Ac and Cry2 A genes were evaluated in the leaves of 10 genotypes(2 parents and 8 Fhybrids)at 30 days after sowing(DAS),while samples of leaves,bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS.The Fhybrids were developed through reciprocal crosses between two Bt(CKC-1,CKC-2)and two non-Bt(MNH-786,FH-942)parents.The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay(ELISA).The results showed that the MNH786 xCKC-1 hybrid had the highest concentrations of Cry1 Ac gene at30 DAS(3.08μg g^(-1))and 110 DAS(1.01μg g^(-1))in leaves.In contrast,the CKC-2 xMNH-786 hybrid showed the lowest concentrations of Cry1 Ac gene at 30 DAS(2.30μg g^(-1))and 110 DAS(0.86μg g^(-1)).The Fhybrid FH-942×CKC-2 showed the highest concentrations of Cry2 A gene at 30 DAS(8.39μg g^(-1))and 110 DAS(7.74μg g^(-1))in leaves,while the CKC-1 xMNH-786 hybrid expressed the lowest concentrations of Cry2 A gene at 30 DAS(7.10μg g^(-1))and 110 DAS(8.31μg g^(-1)).A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS,whereas the lowest concentrations were observed at 110 DAS for both genes in leaves.When the expression pattern was compared between various plant parts in genotype CKC-2,it was found that leaves had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))at 70 DAS,followed by bolls(Cry1 Ac(1.66μg g^(-1))and Cry2 A(8.15μg g^(-1)))and flowers(Cry1 Ac(1.07μg g^(-1))and Cry2 A(7.99μg g^(-1))).The genotype CKC-2 had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))in the upper canopy but less accumulation(2.66μg g^(-1)of Cry1 Ac,8.09μg g^(-1)of Cry2 A)in the lower canopy at 70 DAS.Similarly,at 110 DAS,the expression levels of Cry1 Ac and Cry2 A in upper and lower canopy leaves were 1.52 and 7.92μg 9,and 0.99 and 7.54μg 9,respectively.Hence,the current study demonstrates that different genotypes showed variable expression for both of the Cry1 Ac and Cry2 A genes during plant growth due to different genetic backgrounds.The Cry2 A gene had three-fold higher expression than Cry1 Ac with significant differences in expression in different plant parts.The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1 Ac and Cry2 A for sustainable cotton production worldwide.
文摘A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.
文摘Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard methods of shRNA transgenic (Tg) mice production have not been established. Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. Then high efficiency of Sry gene silencing in Tg mice should induce XY male-to-female sex reversal. An shRNA Tg mouse targeting Sry gene was attempted to be generated by pronuclear microinjection. A low rate (Tg pups/all pups born after microinjection = 2/154 to 7/178) of Tg pups was observed. These Tg mice showed no XY male-to-female sex reversal. The results suggest that exogenous expression of small RNA might exert a negative effect on embryonic development and another approach should be needed for RNAi transgenesis in mice.
文摘Over the last few decades, dengue fever epidemics have increased in frequency and intensity worldwide, making it a major global concern for public health. Its prevention, which is essentially vector-based control, is already being compromised by reports of resistance of the main vector Aedes aegypti to insecticides. To tackle the rapid increase in insecticide resistance and outbreaks, the biological vector control is a promising approach. One of the strategies of this approach is the use of entomopathogenic fungi because of their great efficacy and their eco-friendly aspects. However, some aspects of their use, such as the low efficiency, the high cost of production and the sensitivity to various adverse conditions, need to be addressed for their successful large-scale application. Therefore, innovative technologies based on strains of transgenic fungi with improved biocontrol potentials by genetic engineering are actively pursued. Although these modified mycoinsecticides are acclaimed for their better effectiveness against target insects, the main concern remains their potential adverse effects on the environment and human health. The present review is dedicated to giving an update on recent developments in transgenic entomopathogenic fungi (TEF) for Aedes mosquito control. Future perspectives are also proposed to address the safety concerns related to the release of transgenic entomopathogenic fungi into the environment.
基金the support from the Shenzhen Science and Technology Program, China (KQTD2018041 1143628272)the special funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District, China (PT202101-02)the National Key Research and Development Program of China (2022YFD1700201)。
文摘Chemicals that modify pest behavior are developed to reduce crop damage by altering pest behavior, using specific genes within the olfactory system as molecular targets. The identification of these molecular targets in Bactrocera dorsalis, also known as the functional study of key olfactory genes, relies on CRISPR/Cas9-mediated gene knockout techniques. However, these techniques face limitations when applied to lethal genes. Transgenic technology offers a solution since it enables precise manipulation of gene expression in specific tissues or during certain developmental stages. Consequently, this study developed a piggyBac-mediated transgenic system in B. dorsalis to investigate reporter gene expression in olfactory organs, and assessed the olfactory behavior andantennal electrophysiological responses in transgenic lines. The goal was to assess the potential of this approach for future research on olfactory gene function. A universally expressed housekeeping gene from the BdorActin family was identified using the developmental transcriptome dataset. Its candidate promoter region(BdorActinA3a-1^(P–2k)) was then cloned into the piggyBac plasmid. We subsequently established two stable transgenic lines with specific TTAA insertion sites on chromosomes 4 and 5, consistent with the characteristics of piggyBac transposition. The transgenic strains exhibited essentially normal survival, with hatchability and adult lifespan unaffected, althoughthere were slight reductions in the emergence rate and oviposition capacity. The fluorescent reporter has been successfully expressed in olfactory-related organs, such as the antennae, proboscis, maxillary palp, legs, external genitalia, and brain. The antennal electrophysiological responses to representative chemicals in the transgenic lines were consistent with those of the wild type. However, some olfactory-related behaviors, such as pheromone response and mating, were significantly affected in the transgenic lines. These findings suggest that our system could potentially be applied in future olfactory research, such as driving the expression of exogenous elements that are effective in olfactory organs. However, caution is advised regarding its impact when applied to some olfactoryrelated behavioral phenotypes.
基金M/s.RASI Seeds Pvt.Ltd.,Attur,Tamil Nadu,India for their generous financial assistance in setting up a MAS study in cotton for genetic improvement of sucking pest resistance.
文摘In addition to the negative consequences of climate change,sucking pest complexes severely limited cotton yields in the recent past.Although the damage caused by bollworms was much reduced by utilizing Bt cotton,the emergence of sucking pests(such as aphids,thrips,and whiteflies)poses a serious threat to cotton production,as they reduce lint yield by 40%–60%finally.Additionally,these pests also caused yield losses by spreading viral diseases.Promoting innovative and thorough control methods is necessary to counter the threat posed by these sucking pests.Such initiatives necessitate a multifaceted strategy that combines next-generation breeding technology and pest management techniques to produce novel cotton cultivars that are resistant to sucking pests.The discovery of novel genes and regulatory factors linked to cotton’s resistance to sucking pests will be possible by the combination of next-generation breeding technologies and omics approaches and employing those tools on special resistant donors.Continuous research aimed at understanding the genetic basis of insect resistance and improving integrated pest management(IPM)techniques is crucial to the sustainability and resilience of cotton cropping systems.To this end,a sustainable and viable strategy to protect cotton fields from sucking pests is outlined.
文摘Background Transgenic research in crops involves using genetic engineering techniques to introduce specific genes of interest from other organisms,or even entirely new genes into plant genomes to create crops with desirable traits that wouldn’t be possible through conventional breeding methods.Transgenic crops have been developed for various traits globally.Whitefly,Bemisia tabaci(Gennadius)is one of the major sucking pests of cotton that cause significant damage to the cotton production.To combat whitefly infestations,researchers have developed four transgenic cotton lines expressing the fern protein.And those transgenic lines need to be evaluated for their performance against the target pest—whitefly.The evaluation was designed as controlled trials in polyhouse or muslin cloth cages under open-choice and no-choice conditions by comparing four transgenic cotton lines(A,B,C,and D)with three control groups,including untransformed cotton plants with a same genetic background of the transgenic line,conventionally bred whitefly-resistant cotton,and whitefly-susceptible cotton.In order to study the generational effect,the evaluation also involved studies on whitefly development in laboratory,muslin cloth cage,and polyhouse conditions.Results Both open-choice and no-choice experiments had shown that all the four transgenic cotton lines(A,B,C,and D)expressing the fern protein reduced adult whitefly numbers significantly compared with the control lines,except for the no-choice conditions in 2021,where the transgenic line C was non-significant different from the resistant control line.Notably,the nymphal population on the resistant control line was relatively low and nonsignificant different from the transgenic line C in 2021;and the transgenic lines A and C in 2022 under open-choice conditions.Under no-choice condition,the nymphal counts in the resistant control line was non-significant different from transgenic lines C and D in 2021;and transgenic line D in 2022.All transgenic lines showed significant decrease in egg hatching in 2021 and nymphal development in 2022,except for the transgenic line C which had no significant different in the nymphal development comparing with non-transgenic control lines in 2022.Adult emergence rates in both years of evaluation showed significant decrease in transgenic lines A and B comparing with the control lines.Additionally,the results showed a significant reduction in cotton leaf curl disease and sooty mold development in all the four transgenic lines compared with susceptible control under open-choice conditions,indicating potential benefits of transgenic lines beyond direct effect on whitefly control.Furthermore,the research explored the generational effects of the fern protein on whitefly which revealed the lowest fecundity in the transgenic line C across F0,F1 and F3 generations,lower egg hatching in F1 and F2 generations in transgenic lines A and B,shorter nymphal duration in F1 and F2 generations in transgenic line B,and the least total adult emergence in the transgenic line C in F0 and F3 generations.Conclusions These findings suggest that the transgenic cotton lines expressing fern protein disrupts whitefly populations and the life cycle to a certain extent.However,results are not consistent over generations and years of study,indicating these transgenic lines were not superior over control lines and need to be improved in future breeding.
基金supported by grants from the Major Project on Agricultural Bio-breeding of China(2023ZD04026)National Natural Science Foundation of China(31872866 and 32372124)+2 种基金China Postdoctoral Science Foundation(2022M721101)National Natural Science Foundation of Hunan(2023JJ40132)Natural Science Foundation of Chongqing(CSTB2023NSCQ-MSX0542).
文摘Wheat(Triticum aestivum L.)is a highly valued cereal crop produced and consumed globally,particularly in arid or semi-arid regions(Zhou et al.,2020;Mao et al.,2023).However,its production is increasingly threatened by the rising incidence of drought events associated with climate change.Arid regions are especially susceptible to these droughts,which are intensifying in both severity and frequency(Eckardt et al.,2023;Mao et al.,2023;Yang and Qin,2023).As of a 2022 report,more than 92%of wheat-producing regions are estimated to experience one or more drought/heatwave events in each growing season.Furthermore,the duration and frequency of these combined stress events have increased by approximately 28%over the past four decades(He et al.,2022).To address this challenge,wheat breeding programs have allocated substantial and research efforts to developing elite,stress tolerant lines.This initiative is large part by rapid innovation in transgenic and genome editing strategies(Hu and Xiong,2014;Gao et al.,2021.
基金National Key Research and Development Program of China,Grant/Award Number:2023YFF1000204National Natural Science Foundation of China,Grant/Award Number:32172715,32230105 and 32330103+2 种基金The Innovative Project of State Key Laboratory of Animal Biotech Breeding,Grant/Award Number:2024SKLAB1-2/9Chinese Universities Scientific Fund,Grant/Award Number:2024TC167The 2115 Talent Development Program of China Agricultural University。
文摘Background : Traditional DNA microinjection methods used in mammals are difficult to apply to avian species due to their unique reproductive characteristics. Genetic manipulation in chickens, particularly involving immature follicles within living ovaries, has not been extensively explored. This study seeks to establish an efficient method for generating transgenic chickens through ovarian injection, potentially bypassing the challenges associated with primordial germ cell (PGC) manipulation and fertilized egg microinjection. Methods : Hens were anesthetized and underwent a surgical procedure to access the ovary for DNA injection into immature follicles. The study used liposomes to deliver GFP- expressing plasmids at various dosages. After injection, hens recovered, and their eggs were fertilized through artificial insemination. Results : Transgenic chickens were successfully generated in one generation without needing G0 founders. The injection of 20 μg plasmid yielded the highest transgenic efficiency at 12.1%. GFP- positive embryos were confirmed through microscopy, and successful transgene expression was validated at the tissue level using immunostaining. TERT and GFP elements introduced in the G1 generation resulted in 4.1% positive transgene rates, as confirmed by PCR and Southern blotting. Conclusion : This ovarian injection method offers a promising alternative for avian genetic manipulation, bypassing complex PGC procedures and enabling direct generation of G1 transgenic chickens. This technique simplifies the transgenic process for chickens and has the potential to be adapted for other avian species, especially those without established PGCs culture systems.
基金supported by the Key Project of the National Natural Science Foundation of China(No.42230711)。
文摘Aluminium(Al)toxicity is one of the key factors limiting crop output in acidic soils,but until now little has been known about how Al is regulated transcriptionally in plants.This study identified Arabidopsis NAC transcription factor ANAC050 in the regulation of Al tolerance.ANAC050 was located in the nucleus and displayed constitutive expression in the silique,flower,leaf,stem,and root,despite the fact that Al stress decreased its expression and protein accumulation.When compared with the Columbia ecotype wild-type,anac050 mutants that lacked function of ANAC050 exhibited Al sensitivity phenotype,while transgenic lines that overexpressed ANAC050 showed an Al-resistant phenotype,indicating the favorable influence of ANAC050 on preserving Al tolerance in plants.Further analysis indicated that anac050 mutants accumulated more Al in roots,implying that ANAC050 may confer a potential operation of an Al exclusion mechanism.Interestingly,anac050 mutants had down-regulated the expression of the genes encoding MULTIDRUG AND TOXIC COMPOUND EXTRUSION(MATE)and AL-ACTIVATED MALATE TRANSPORTER(ALMT1),which were involved in the secretion of citrate and malate,even though there was no evidence of a direct interaction between them,suggesting ANAC050 may mediate the secretion of citrate and malate indirectly.Together with the decreased hemicellulose content,lower Al content was also discovered in root cell walls and hemicelluloses of anac050 mutants,pointing to a potential interaction between ANAC017 and XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE(XTH).Although there was no evidence of a direct interaction between ANAC050 and XTH31,it is worth mentioning that the expression of XTH31,which is essential for xyloglucan modification,was down-regulated in anac050 mutants irrespective of the amount of Al given.In conclusion,our findings showed that ANAC050 contributed to Al resistance by indirect control of the release of organic acids and the accumulation of cell wall hemicelluloses.
基金supported by Key Project of Jiangsu Province’s Administration of Traditional Chinese Medicine(ZD202203)Jiangsu Province’s Innovation Program(JSSCTD202142)a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions(traditional Chinese medicine).
文摘Traditional Chinese medicine(TCM)can help prevent or treat diseases;however,there are few studies on the active substances of TCM.For example,Lycium barbarum L.has been proven to be effective in treating osteoporosis for thousands of years,but its active substance remains to be unknown.Prompted by the efforts to modernize TCM,the present study focused on the novel active substance of Lycium barbarum L.to reinforce kidney essence to produce bone marrow.Illumina deep sequencing analysis and stemloop polymerase chain reaction(PCR)assay revealed that miR162a,a Lycium barbarum L.-derived microRNA,can pass through the gastrointestinal tract to target the bone marrow in mice.Immunofluorescence staining showed that miR162a was absorbed through systemic RNA interference defective transmembrane family member 1(SIDT1)in the stomach.Bioinformatics prediction and luciferase reporter assay identified that miR162a targeted nuclear receptor corepressor(NcoR).Alizarin red staining and micro-computed tomography(microCT)confirmed that miR162a promoted osteogenic differentiation in bone marrow mesenchymal stem cells,zebrafish,and a mouse model of osteoporosis.In addition,transgenic Nicotiana benthamiana(N.benthamiana)leaves overexpressing miR162a were developed by agrobacterium infiltration method.microCT and tartrate-resistant acid phosphatase staining confirmed that transgenic N.benthamiana leaves effectively protected against osteoporosis in mice.Our study mechanistically explains how Lycium barbarum L.improves osteoporosis and supports that Lycium barbarum L.reinforces kidney essence,thereby strengthening the bone.miR162a expressed by transgenic plants may represent a novel and safe treatment for human osteoporosis.
基金supported by grants from the National Key Research and Development Program of China(2019YFA0802704)the National Natural Science Foundation of China(31771620)+2 种基金the Natural Science Foundation of Chongqing,China(CSTB2022NSCQMSX1424)Research Startup Fund of Southwest University(SWU117064)Open Research Fund of National Health Commission Key Laboratory of Birth Defects Prevention&Henan Key Laboratory of Population Defects Prevention(ZD202302)。
文摘Cilia are indispensable for organ development and function,and their dysfunction causes a range of syndromic diseases known as ciliopathies,including obesity,cystic kidney disease,situs inversus,and male infertility(Reiter and Leroux,2017;Wallmeier et al.,2020).To date,over 180 ciliopathy-associated genes have been identified(Reiter and Leroux,2017),yet the underlying mechanisms remain poorly understood.
基金supported by grants from the Jiangxi Provincial Natural Science Foundation,No.20242BAB26134(to XF)the National Natural Science Foundation of China,Nos.82060638(to TC),82060222(to XF),82460237(to XF)+1 种基金the Major Disciplines of Academic and Technical Leaders Project of Jiangxi Province,Nos.20194BCJ22032(to TC),20213BCJL22049(to XF)Science and Technology Plan of Jiangxi Health Planning Committee,No.202210390(to XF).
文摘Parkinson’s disease is characterized by synucleinopathy-associated neurodegeneration.Previous studies have shown that glucagon-like peptide-1(GLP-1)has beneficial effects in a mouse model of Parkinson’s disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.However,the effect of GLP-1 on intrinsic synuclein malfunction remains unclear.In this study,we investigated the effect of Lactococcus lactis MG1363-pMG36e-GLP-1 on parkinsonism in SncaA53T transgenic mice and explored the underlying mechanisms.Our data showed that Lactococcus lactis MG1363-pMG36e-GLP-1 inhibited dopaminergic neuronal death,reduced pathological aggregation ofα-synuclein,and decreased movement disorders in SncaA53T transgenic mice.Furthermore,Lactococcus lactis MG1363-pMG36e-GLP-1 downregulated lipopolysaccharide-related inflammation,reduced cerebral activation of microglia and astrocytes,and promoted cell survival via the GLP-1 receptor/PI3K/Akt pathway in the substantia nigra.Additionally,Lactococcus lactis MG1363-pMG36e-GLP-1 decreased serum levels of pro-inflammatory molecules including lipopolysaccharide,lipopolysaccharide binding protein,interleukin-1β,and interleukin-6.Gut histopathology and western blotting further revealed that Lactococcus lactis MG1363-pMG36e-GLP-1 increased the expression of gut integrity-related proteins and reduced lipopolysaccharide-related inflammation by reversing gut dysbiosis in SncaA53T transgenic mice.Our findings showed that the beneficial effect of Lactococcus lactis MG1363-pMG36e-GLP-1 on parkinsonism traits in SncaA53T transgenic mice is mediated by microglial polarization and the reversal of dysbiosis.Collectively,our findings suggest that Lactococcus lactis MG1363-pMG36e-GLP-1 is a promising therapeutic agent for the treatment of Parkinson’s disease.
基金supported by the Development Plan of the State Key Fun-damental Research of China (Grant Nos. 2007CB109205, 2007CB109206)the National Natural Science Foundation of China (Grant No. 30930069)
文摘It has been more than 20 years since the first batch of transgenic fish was produced. Five stable germ-line transmitted growth hormone (GH) transgenic fish lines have been generated. This paper reviews the mechanisms of integration and gene targeting of the transgene, as well as the viability, reproduction and transgenic approaches for the reproductive containment of GH-transgenic fish. Further, we propose that it should be necessary to do the following studies, in particularly, of the breeding of transgenic fish: to assess the fitness of transgenic fish in an aqueous environment with a large space and a complex structure; and to develop a controllable on-off strategy of reproduction in transgenic fish.
基金the National Natural Science Foundation of China (Grant No. 39730290).
文摘To gain information on the integration pattern of pMThGH-transgene, 50 transgenes were recovered from F4 generation of pMThGH transgenic common carp (Cyprinus carpio L.) and 33 recovered genes were analyzed. The restriction maps of these recovered genes were constructed by digestion with five kinds of enzymes. These transgenes can be classified into 4 types according to their restriction maps. Only one type of transgenes maintains its original molecular form, whereas the other three types are very different from the original one and vary each other on both molecular weight and restriction maps. This implies that the sequences of most transgenes have been deleted and/or rearranged during integration and inheritance. The results of PCR amplification and Southern blot hybridization indicate that MThGH in Type I transgene keeps intact but most of its sequence has been deleted in other three types. All these results suggest that transgenes in F4 generation of transgenic carp are highly polymorphic. Two DNA
基金supported by the National Science and Technology Innovation 2030 Major Projects(2021ZD0202200)National Natural Science Foundation of China(32171090,81970264)+1 种基金Shanghai Science and Technology Commission(21ZR1482600)2023 Youth Innovation Promotion Association CAS。
文摘Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes.However,their activity markedly diminishes with payloads exceeding 11 kb.Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs,improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics,metabolic engineering,and transgenic animal production.In this study,we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer(QBI SP163,ST)and enhanced the nuclear targeting ability using the nuclear localization protein H2B(SHT).The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures(H1299),comparable to the well-established super PiggyBac system.Furthermore,mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads(8 kb,14 kb,and 24 kb)into zebrafish(Danio rerio).This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.
基金supported by the National Key Research and Development Program of China (2021YFA0805300,2021YFA0805200)National Natural Science Foundation of China (32170981,82371874,82394422,82171244,82071421,82271902)+1 种基金Guangzhou Key Research Program on Brain Science (202007030008)Department of Science and Technology of Guangdong Province (2021ZT09Y007,2020B121201006,2018B030337001)。
文摘Huntington'sdisease(HD)isahereditary neurodegenerative disorder for which there is currently no effectivetreatmentavailable.Consequently,the development of appropriate disease models is critical to thoroughly investigate disease progression.The genetic basis of HD involves the abnormal expansion of CAG repeats in the huntingtin(HTT)gene,leading to the expansion of a polyglutamine repeat in the HTT protein.Mutant HTT carrying the expanded polyglutamine repeat undergoes misfolding and forms aggregates in the brain,which precipitate selective neuronal loss in specific brain regions.Animal models play an important role in elucidating the pathogenesis of neurodegenerative disorders such as HD and in identifying potential therapeutic targets.Due to the marked species differences between rodents and larger animals,substantial efforts have been directed toward establishing large animal models for HD research.These models are pivotal for advancing the discovery of novel therapeutic targets,enhancing effective drug delivery methods,and improving treatment outcomes.We have explored the advantages of utilizing large animal models,particularly pigs,in previous reviews.Since then,however,significant progress has been made in developing more sophisticated animal models that faithfully replicate the typical pathology of HD.In the current review,we provide a comprehensive overview of large animal models of HD,incorporating recent findings regarding the establishment of HD knock-in(KI)pigs and their genetic therapy.We also explore the utilization of large animal models in HD research,with a focus on sheep,non-human primates(NHPs),and pigs.Our objective is to provide valuable insights into the application of these large animal models for the investigation and treatment of neurodegenerative disorders.
基金the Projects of International Cooperation National Key R&D Program of China(Grant No.2022YFE0108300)the National Key Research and Development Program of China(Grant No.2022YFF1003000)the National Natural Science Foundation of China(Grant Nos.32372682,32272747,32072585,32072568).
文摘Selenocysteine methyltransferase(SMT)is a key enzyme involved in the Se metabolism pathway,and it is responsible for the catalysis of Se-methylselenocysteine(SeMSC)compound formation.Previous studies showed that selenium treatment activated SMT expression and promoted the accumulation of glucosinolates(GSLs)and sulforaphane,but the roles and functional mechanisms of SMT in mediating GSLs and sulforaphane synthesis remain unclear.In this study,we identified the BoSMT gene in broccoli and uncovered its roles in mediating GSLs biosynthesis.Transgenic assays revealed that BoSMT is involved in SeMSC biosynthesis in broccoli.More importantly,the contents of GSLs and sulforaphane were significantly increased in the BoSMT-overexpressing broccoli lines but decreased in the knockdown lines,suggesting that BoSMT played a positive role in regulating GSLs and sulforaphane synthesis.Further evidence indicated that BoSMT-mediated overaccumulation of GSLs and sulforaphane might be due to the increase in the endogenous SeMSC content.Compared with the mock(water)treatment,selenite-induced significantly increases of the SeMSC content in the BoSMT-knockdown plants partially compensated the phenotype of GSLs and sulforaphane loss.Compared with the mock treatment,exogenous SeMSC treatment significantly increased the contents of GSL and sulforaphane and activated GSL synthesis-related gene expression,suggesting that SeMSC acted as a positive regulator for GSL and sulforaphane production.Our findings provided novel insights into selenium-mediated GSLs and sulforaphane accumulation.The genetic manipulation of BoSMT might be a useful strategy for improving the dietary nutritional values of broccoli.
基金funded by National Natural Science Foundation of China (Grant No.32072575)Postgraduate Research & Practice Innovation Program of Jiangsu Province (Grant No.KYCX20_0588)National Vegetable Industry Technology System (Grant No.CARS-23-A16)。
文摘Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.
