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
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
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
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
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
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
OBJECTIVE:To discuss the influence of Sailuotong(塞络通,SLT)on the Neurovascular Unit(NVUs)of amyloid precursor protein(APP)/presenilin-1(PS1)mice and evaluate the role of gas supplementation in activating blood circu...OBJECTIVE:To discuss the influence of Sailuotong(塞络通,SLT)on the Neurovascular Unit(NVUs)of amyloid precursor protein(APP)/presenilin-1(PS1)mice and evaluate the role of gas supplementation in activating blood circulation during the progression of Alzheimer's disease(AD).METHODS:The mice were allocated into the following nine groups:(a)the C57 Black(C57BL)sham-operated group(control group),(b)ischaemic treatment in C57BL mice(the C57 ischaemic group),(c)the APP/PS1 sham surgery group(APP/PS1 model group),(d)ischaemic treatment in APP/PS1 mice(APP/PS1 ischaemic group),(e)C57BL mice treated with aspirin following ischaemic treatment(C57BL ischaemic+aspirin group),(f)C57BL mice treated with SLT following ischaemic treatment(C57BL ischaemic+SLT group),(g)APP/PS1 mice treated with SLT(APP/PS1+SLT group),(h)APP/PS1 mice treated with donepezil hydrochloride following ischaemic treatment(APP/PS1 ischaemic+donepezil hydrochloride group)and(i)APP/PS1 mice treated with SLT following ischaemic treatment(APP/PS1 ischaemic+SLT group).The ischaemic model was established by operating on the bilateral common carotid arteries and creating a microembolism.The Morris water maze and step-down tests were used to detect the spatial behaviour and memory ability of mice.The hippocampus of each mouse was observed by haematoxylin and eosin(HE)and Congo red staining.The ultrastructure of NVUs in each group was observed by electron microscopy,and various biochemical indicators were detected by enzymelinked immunosorbent assay(ELISA).The protein expression level was detected by Western blot.The mRNA expression was detected by quantitative real-time polymerase chain reaction(qRT-PCR).RESULTS:The results of the Morris water maze and step-down tests showed that ischemia reduced learning and memory in the mice,which were restored by SLT.The results of HE staining showed that SLT restored the pathological changes of the NVUs.The Congo red staining results revealed that SLT also improved the scattered orange-red sediments in the upper cortex and hippocampus of the APP/PS1 and APP/PS1 ischaemic mice.Furthermore,SLT significantly reduced the content of Aβ,improved the vascular endothelium and repaired the mitochondrial structures.The ELISA detection,western blot detection and qRT-PCR showed that SLT significantly increased the vascular endothelial growth factor(VEGF),angiopoietin and basic fibroblast growth factor,as well as the levels of gene and protein expression of low-density lipoprotein receptor-related protein-1(LRP-1)and VEGF in brain tissue.CONCLUSIONS:By increasing the expression of VEGF,SLT can promote vascular proliferation,up-regulate the expression of LRP-1,promote the clearance of Aβand improve the cognitive impairment of APP/PS1 mice.These results confirm that SLT can improve AD by promoting vascular proliferation and Aβclearance to protect the function of NVUs.展开更多
Background Chitinase is an enzyme that hydrolyzes chitin,a major component of the exoskeleton of insects,including plant pests like whiteflies.The present study aimed to investigate the expression of chemically synthe...Background Chitinase is an enzyme that hydrolyzes chitin,a major component of the exoskeleton of insects,including plant pests like whiteflies.The present study aimed to investigate the expression of chemically synthesized barley ch1 and chi2 genes in cotton(Gossypium hirsutum)through Agrobacterium-mediated transformation.Fifty-five putative transgenic cotton plants were obtained,out of which fifteen plants successfully survived and were shifted to the field.Using gene-specific primers,amplification of 447 bp and 401 bp fragments confirmed the presence of the ch1 and chi2 genes in five transgenic cotton plants of the T0 generation.These five plants were further evalu-ated for their mRNA expression levels.The T0 transgenic cotton plants with the highest mRNA expression level and better yield performance in field,were selected to raise their subsequent progenies.Results The T1 cotton plants showed the highest mRNA expression levels of 3.5-fold in P10(2)for the ch1 gene and 3.7-fold in P2(1)for the chi2 gene.Fluorescent in situ hybridization(FISH)confirmed a single copy number of ch1 and chi2(hemizygous)on chromosome no.6.Furthermore,the efficacy of transgenes on whitefly was evaluated through an insect bioassay,where after 96 h of infestation,mortality rates of whitefly were calculated to be 78%–80%in transgenic cotton plants.The number of eggs on transgenic cotton plants were calculated to be 0.1%–0.12 per plant compared with the non-transgenic plants where egg number was calculated to be 0.90–1.00 per plant.Conclusion Based on these findings,it can be concluded that the chemically synthesized barley chitinase genes(ch1 and chi2)have the potential to be effective against insects with chitin exoskeletons,including whiteflies.The transgenic cotton plants expressing these genes showed increased resistance to whiteflies,resulting in reduced egg numbers and higher mortality rates.展开更多
One of the most prevalent disorders that cause blindness worldwide is cataract,and its essence is the visual disorder caused by the opacity of the lens.The significant degree of variation in cataracts and the fact tha...One of the most prevalent disorders that cause blindness worldwide is cataract,and its essence is the visual disorder caused by the opacity of the lens.The significant degree of variation in cataracts and the fact that a variety of factors can impact a patient’s lens transparency make it especially crucial to investigate the pathogenesis of cataracts at the molecular level.It has been found that more than 60 genes are linked to the formation of cataracts,and the construction of a transgenic mouse model of cataract similar to the selection of human lens clouding due to a variety of causes has become an important means of studying the pathogenesis of cataract.Therefore,the research on the application of transgenic mice to the molecular pathogenesis of cataracts will be the main topic of this review of the literature.展开更多
[Objective] The aim of the research was to analyze the resistance of binary insect-resistant transgenic soybean to Heliothis viriplaca.[Method]In this experiment, resistance analysis of the stabilized binary insect-re...[Objective] The aim of the research was to analyze the resistance of binary insect-resistant transgenic soybean to Heliothis viriplaca.[Method]In this experiment, resistance analysis of the stabilized binary insect-resistant transgenic soybean to Heliothis viriplaca was conducted in lab and in field conditions.[Result] The results indicated that the leaves of insect-resistant transgenic soybeans T5-150 and T5-195 showed lighter damage than those of non-transgenic soybeans. Meanwhile, the Heliothis viriplaca larvae fed on leaves of these two transgenic soybeans were characterized by less leaf consumption, shortening survival day, slower development and less pupation.[Conclusion]It was concluded that insect-resistance of transgenic soybean to Heliothis viriplaca was increased dramatically and the research provided a reference for selecting binary insect-resistant transgenic soybean to Heliothis viriplaca.展开更多
Lysine-rich protein gene (lys) was cloned from Psophocarpus tetragonolobus (L.) DC. A plant expression plasmid was constructed and lys gene was under the control of maize ubiquitin promoter which is the highest effici...Lysine-rich protein gene (lys) was cloned from Psophocarpus tetragonolobus (L.) DC. A plant expression plasmid was constructed and lys gene was under the control of maize ubiquitin promoter which is the highest efficient monocotyledon promoter. The plasmid was introduced into rice embryogenic calli by microprojectile bombardment. The regenerated fertile plants were obtained by effective selection for hygromycin B resistance. Genomic PCR and Southern blotting analyses showed that the lys gene has been integrated into rice genome. Simultaneously, the results of GUS histochemical assay demonstrated that gus report gene is also expressed in leaves, stems and roots of the transgenic rice plants. Data analysis showed that lysine content in most of the 11 transgenic plants is differently improved, and in one of them increased by 16.04%.展开更多
The seed_specific phaseolin promoter (Ph/P) was fused to an ipt gene, then was cloned to a plant expression vector containing a gus gene driven by a 35S promoter. Cotton (Gossypium hirsutum L.) plants were tr...The seed_specific phaseolin promoter (Ph/P) was fused to an ipt gene, then was cloned to a plant expression vector containing a gus gene driven by a 35S promoter. Cotton (Gossypium hirsutum L.) plants were transformed through pollen tube pathway methods. After seed germination, histochemical staining of the roots demonstrated that 32 GUS positive plants were obtained and three of which contained the chimeric Ph/P_ ipt transgene as confirmed by PCR analysis. An immunosorbent assay showed that two of the three transgenic cotton lines contained higher levels of zeatin equivalents in seeds than the control. Seedling development of these two transgenic lines differed from the control in a reduction of the shoot growth, showing a stunted phenotype as expected, but a surprisingly developed root system with a 3-4 fold fast_growing lateral roots. In addition, fibers (seed_hairs) of the two transgenic cotton lines were considerably shorter than those of the control. These results indicate that genetic engineering may be used to manipulate the development of cotton plants, particularly cotton fibers.展开更多
Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is...Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is catalyzed by choline monooxygenase (CMO). In this study, the full-length CMO cDNA (1 820 bp) was cloned from halophyte Suaeda liaotungensis Kitag by RT-PCR and RACE. It included a 123 bp 5' UTR, a 368 bp 3' UTR and a 1 329 bp open reading frame encoding a 442-amino-acid polypeptide with 77%, 72% and 74% sequence identity compared to CMOs from spinach, sugar beet and Atriplex hortensis, respectively. The CMO open reading frame (ORF) was cloned and the plant expression vector pBI121-CMO was constructed. It was transferred into tobacco ( Nicotiana tabacum L. ev. 89) via Agrobacterium mediation. PCR and Southern blotting analysis showed that the CMO gene was integrated into tobacco genome. Transgenic tobacco plants contained higher amount of betaine than that of control plants and were able to survive on MS medium containing 250 mmol/L NaCl. Relative electronic conductivity demonstrated less membrane damage in transgenic plants as in the wild type.展开更多
The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r）...The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r） gene and enhanced green fluorescent protein （EGFP） gene linked through an inner ribosomal entry site （IRES） sequence directed by a Cytomegalovirus （CMV） promoter, was used for enrichment and selection of the transgenic cells and preimplantation embryos. Transgenes were introduced into bovine fetal fibroblast cells （BFF） cultured in vitro through electroporation （900 V/cm, 5 ms）. Transgenic bovine fibroblast cells （TBF） were enriched through addition of G418 in culture medium （800 μg/mL）. Before being used as a nuclear donor, the TBF cells were either cultured in normal conditions （10% FBS） or treated with serum starvation （0.5% FBS for 2-4 days） followed by 10 hours recovery for G1 phase synchronization. Transgenic cloned embryos were produced through GFP-expressing cell selection and SCNT. The results were the percentage of blastocyst development following SCNT was lower using TBF than BFF cells （23.2% VS 35.2%, P 〈 0.05）. No difference in the percentage of cloned blastocysts between the two groups of transgenic nuclear donor of normal and starvation cultures were observed （23.2% VS 18.9%, P 〉 0.05）. Two to four GFP-expressing blastocysts were transferred into the uterus of each synchronised recipient. One pregnancy from of seven recipients （21 embryos） was confirmed by rectum palpation 60 days after embryo transfer and one recipient has given birth to a calf at term. PCR and DNA sequencing analysis confirmed that the calf was produced using human proinsulin transgenic animal.展开更多
A gene sequence coding for the precursor of Galanthus nivalis agglutinin (GNA) was modified by site-directed mutagenesis to change very low usage bias codons to higher usage bias ones for improvement of the gene expre...A gene sequence coding for the precursor of Galanthus nivalis agglutinin (GNA) was modified by site-directed mutagenesis to change very low usage bias codons to higher usage bias ones for improvement of the gene expression in transgenic tobacco (Nicotiana tabacum L.) plants. Results from Western blot analysis of some of the transgenic tobacco plants showed that the expression level of GNA in plants transformed with the modified gene GNA34m reached 0.25% of total soluble proteins, while that of the GNA34 gene transgenic plants was 0.17%. Since the GNA expression level increased, the aphid resistance of GNA34m transgenic plants were also enhanced significantly as judged by a 71.0% aphid population inhibition in insect bioassay of GNA34m transformed plants and 63.7% for the plants transformed with the natural GNA34 gene.展开更多
[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer techn...[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.展开更多
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.
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 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 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.
基金National Natural Science Foundation of China(81503450):Experimental study on the treatment of transgenic mice with Alzheimer's disease by protecting neurovascular unit by supplementing Qi and activating blood circulation investigate。
文摘OBJECTIVE:To discuss the influence of Sailuotong(塞络通,SLT)on the Neurovascular Unit(NVUs)of amyloid precursor protein(APP)/presenilin-1(PS1)mice and evaluate the role of gas supplementation in activating blood circulation during the progression of Alzheimer's disease(AD).METHODS:The mice were allocated into the following nine groups:(a)the C57 Black(C57BL)sham-operated group(control group),(b)ischaemic treatment in C57BL mice(the C57 ischaemic group),(c)the APP/PS1 sham surgery group(APP/PS1 model group),(d)ischaemic treatment in APP/PS1 mice(APP/PS1 ischaemic group),(e)C57BL mice treated with aspirin following ischaemic treatment(C57BL ischaemic+aspirin group),(f)C57BL mice treated with SLT following ischaemic treatment(C57BL ischaemic+SLT group),(g)APP/PS1 mice treated with SLT(APP/PS1+SLT group),(h)APP/PS1 mice treated with donepezil hydrochloride following ischaemic treatment(APP/PS1 ischaemic+donepezil hydrochloride group)and(i)APP/PS1 mice treated with SLT following ischaemic treatment(APP/PS1 ischaemic+SLT group).The ischaemic model was established by operating on the bilateral common carotid arteries and creating a microembolism.The Morris water maze and step-down tests were used to detect the spatial behaviour and memory ability of mice.The hippocampus of each mouse was observed by haematoxylin and eosin(HE)and Congo red staining.The ultrastructure of NVUs in each group was observed by electron microscopy,and various biochemical indicators were detected by enzymelinked immunosorbent assay(ELISA).The protein expression level was detected by Western blot.The mRNA expression was detected by quantitative real-time polymerase chain reaction(qRT-PCR).RESULTS:The results of the Morris water maze and step-down tests showed that ischemia reduced learning and memory in the mice,which were restored by SLT.The results of HE staining showed that SLT restored the pathological changes of the NVUs.The Congo red staining results revealed that SLT also improved the scattered orange-red sediments in the upper cortex and hippocampus of the APP/PS1 and APP/PS1 ischaemic mice.Furthermore,SLT significantly reduced the content of Aβ,improved the vascular endothelium and repaired the mitochondrial structures.The ELISA detection,western blot detection and qRT-PCR showed that SLT significantly increased the vascular endothelial growth factor(VEGF),angiopoietin and basic fibroblast growth factor,as well as the levels of gene and protein expression of low-density lipoprotein receptor-related protein-1(LRP-1)and VEGF in brain tissue.CONCLUSIONS:By increasing the expression of VEGF,SLT can promote vascular proliferation,up-regulate the expression of LRP-1,promote the clearance of Aβand improve the cognitive impairment of APP/PS1 mice.These results confirm that SLT can improve AD by promoting vascular proliferation and Aβclearance to protect the function of NVUs.
文摘Background Chitinase is an enzyme that hydrolyzes chitin,a major component of the exoskeleton of insects,including plant pests like whiteflies.The present study aimed to investigate the expression of chemically synthesized barley ch1 and chi2 genes in cotton(Gossypium hirsutum)through Agrobacterium-mediated transformation.Fifty-five putative transgenic cotton plants were obtained,out of which fifteen plants successfully survived and were shifted to the field.Using gene-specific primers,amplification of 447 bp and 401 bp fragments confirmed the presence of the ch1 and chi2 genes in five transgenic cotton plants of the T0 generation.These five plants were further evalu-ated for their mRNA expression levels.The T0 transgenic cotton plants with the highest mRNA expression level and better yield performance in field,were selected to raise their subsequent progenies.Results The T1 cotton plants showed the highest mRNA expression levels of 3.5-fold in P10(2)for the ch1 gene and 3.7-fold in P2(1)for the chi2 gene.Fluorescent in situ hybridization(FISH)confirmed a single copy number of ch1 and chi2(hemizygous)on chromosome no.6.Furthermore,the efficacy of transgenes on whitefly was evaluated through an insect bioassay,where after 96 h of infestation,mortality rates of whitefly were calculated to be 78%–80%in transgenic cotton plants.The number of eggs on transgenic cotton plants were calculated to be 0.1%–0.12 per plant compared with the non-transgenic plants where egg number was calculated to be 0.90–1.00 per plant.Conclusion Based on these findings,it can be concluded that the chemically synthesized barley chitinase genes(ch1 and chi2)have the potential to be effective against insects with chitin exoskeletons,including whiteflies.The transgenic cotton plants expressing these genes showed increased resistance to whiteflies,resulting in reduced egg numbers and higher mortality rates.
基金Supported by the National Natural Science Foundation of China(No.82271070)the Heilongjiang Provincial Undergraduate Colleges and Universities Central to Support the Reform and Development of Local Colleges and Universities(No.2020YQ08)+1 种基金the Natural Science Foundation Project of Heilongjiang Province(Key Project/Outstanding Youth/Joint Guidance,No.LH2021H112)Doctoral Research Fund of Mudanjiang Medical University Affiliated Hongqi Hospital(No.2024-HQBS-03).
文摘One of the most prevalent disorders that cause blindness worldwide is cataract,and its essence is the visual disorder caused by the opacity of the lens.The significant degree of variation in cataracts and the fact that a variety of factors can impact a patient’s lens transparency make it especially crucial to investigate the pathogenesis of cataracts at the molecular level.It has been found that more than 60 genes are linked to the formation of cataracts,and the construction of a transgenic mouse model of cataract similar to the selection of human lens clouding due to a variety of causes has become an important means of studying the pathogenesis of cataract.Therefore,the research on the application of transgenic mice to the molecular pathogenesis of cataracts will be the main topic of this review of the literature.
文摘[Objective] The aim of the research was to analyze the resistance of binary insect-resistant transgenic soybean to Heliothis viriplaca.[Method]In this experiment, resistance analysis of the stabilized binary insect-resistant transgenic soybean to Heliothis viriplaca was conducted in lab and in field conditions.[Result] The results indicated that the leaves of insect-resistant transgenic soybeans T5-150 and T5-195 showed lighter damage than those of non-transgenic soybeans. Meanwhile, the Heliothis viriplaca larvae fed on leaves of these two transgenic soybeans were characterized by less leaf consumption, shortening survival day, slower development and less pupation.[Conclusion]It was concluded that insect-resistance of transgenic soybean to Heliothis viriplaca was increased dramatically and the research provided a reference for selecting binary insect-resistant transgenic soybean to Heliothis viriplaca.
文摘Lysine-rich protein gene (lys) was cloned from Psophocarpus tetragonolobus (L.) DC. A plant expression plasmid was constructed and lys gene was under the control of maize ubiquitin promoter which is the highest efficient monocotyledon promoter. The plasmid was introduced into rice embryogenic calli by microprojectile bombardment. The regenerated fertile plants were obtained by effective selection for hygromycin B resistance. Genomic PCR and Southern blotting analyses showed that the lys gene has been integrated into rice genome. Simultaneously, the results of GUS histochemical assay demonstrated that gus report gene is also expressed in leaves, stems and roots of the transgenic rice plants. Data analysis showed that lysine content in most of the 11 transgenic plants is differently improved, and in one of them increased by 16.04%.
文摘The seed_specific phaseolin promoter (Ph/P) was fused to an ipt gene, then was cloned to a plant expression vector containing a gus gene driven by a 35S promoter. Cotton (Gossypium hirsutum L.) plants were transformed through pollen tube pathway methods. After seed germination, histochemical staining of the roots demonstrated that 32 GUS positive plants were obtained and three of which contained the chimeric Ph/P_ ipt transgene as confirmed by PCR analysis. An immunosorbent assay showed that two of the three transgenic cotton lines contained higher levels of zeatin equivalents in seeds than the control. Seedling development of these two transgenic lines differed from the control in a reduction of the shoot growth, showing a stunted phenotype as expected, but a surprisingly developed root system with a 3-4 fold fast_growing lateral roots. In addition, fibers (seed_hairs) of the two transgenic cotton lines were considerably shorter than those of the control. These results indicate that genetic engineering may be used to manipulate the development of cotton plants, particularly cotton fibers.
文摘Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is catalyzed by choline monooxygenase (CMO). In this study, the full-length CMO cDNA (1 820 bp) was cloned from halophyte Suaeda liaotungensis Kitag by RT-PCR and RACE. It included a 123 bp 5' UTR, a 368 bp 3' UTR and a 1 329 bp open reading frame encoding a 442-amino-acid polypeptide with 77%, 72% and 74% sequence identity compared to CMOs from spinach, sugar beet and Atriplex hortensis, respectively. The CMO open reading frame (ORF) was cloned and the plant expression vector pBI121-CMO was constructed. It was transferred into tobacco ( Nicotiana tabacum L. ev. 89) via Agrobacterium mediation. PCR and Southern blotting analysis showed that the CMO gene was integrated into tobacco genome. Transgenic tobacco plants contained higher amount of betaine than that of control plants and were able to survive on MS medium containing 250 mmol/L NaCl. Relative electronic conductivity demonstrated less membrane damage in transgenic plants as in the wild type.
文摘The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r） gene and enhanced green fluorescent protein （EGFP） gene linked through an inner ribosomal entry site （IRES） sequence directed by a Cytomegalovirus （CMV） promoter, was used for enrichment and selection of the transgenic cells and preimplantation embryos. Transgenes were introduced into bovine fetal fibroblast cells （BFF） cultured in vitro through electroporation （900 V/cm, 5 ms）. Transgenic bovine fibroblast cells （TBF） were enriched through addition of G418 in culture medium （800 μg/mL）. Before being used as a nuclear donor, the TBF cells were either cultured in normal conditions （10% FBS） or treated with serum starvation （0.5% FBS for 2-4 days） followed by 10 hours recovery for G1 phase synchronization. Transgenic cloned embryos were produced through GFP-expressing cell selection and SCNT. The results were the percentage of blastocyst development following SCNT was lower using TBF than BFF cells （23.2% VS 35.2%, P 〈 0.05）. No difference in the percentage of cloned blastocysts between the two groups of transgenic nuclear donor of normal and starvation cultures were observed （23.2% VS 18.9%, P 〉 0.05）. Two to four GFP-expressing blastocysts were transferred into the uterus of each synchronised recipient. One pregnancy from of seven recipients （21 embryos） was confirmed by rectum palpation 60 days after embryo transfer and one recipient has given birth to a calf at term. PCR and DNA sequencing analysis confirmed that the calf was produced using human proinsulin transgenic animal.
文摘A gene sequence coding for the precursor of Galanthus nivalis agglutinin (GNA) was modified by site-directed mutagenesis to change very low usage bias codons to higher usage bias ones for improvement of the gene expression in transgenic tobacco (Nicotiana tabacum L.) plants. Results from Western blot analysis of some of the transgenic tobacco plants showed that the expression level of GNA in plants transformed with the modified gene GNA34m reached 0.25% of total soluble proteins, while that of the GNA34 gene transgenic plants was 0.17%. Since the GNA expression level increased, the aphid resistance of GNA34m transgenic plants were also enhanced significantly as judged by a 71.0% aphid population inhibition in insect bioassay of GNA34m transformed plants and 63.7% for the plants transformed with the natural GNA34 gene.
基金Supported by the National High-tech R&D Program(2004AA213072)the Doctor Fund of Henan University of Science and Technology~~
文摘[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.
