Tetracycline-inducible Expression Systems： New Strategies and Practices in the Transgenic Mouse Modeling

To accurately analyze the function of transgene(s)of interest in transgenic mice,and togenerate credible transgenic animal models for multifarious human diseases to precisely mimic human dis-ease states,it is critical to tightly regulate gene expression in the animals in a conditional manner.The abilityto turn gene expression on or off in the restricted cells or tissues at specific time permits unprecedentedflexibility in dissecting gene functions in health and disease.Pioneering studies in conditional transgene ex-pression have brought about the development of a wide variety of controlled gene expression systems,whichmeet this criterion.Among them,the tetracycline-controlled expression systems(e.g.Tet-off system andTet-on system)have been used extensively in vitro and in vivo.In recent years,some strategies derived fromtetracycline-inducible system alone,as well as the combined use of Tet-based systems and Cre/lox P switch-ing gene expression system,have been newly developed to allow more flexibility for exploring gene functionsin health and disease,and produce credible transgenic animal models for various human diseases.In thisreview these newly developed strategies are discussed.     

The kinetics of IL—4 and IFN—γ gene expression in Mice after Trichosansin immunization

Trichosanthin (TCS) is a potent allergen to mice.According to our previous experiments,it could bring out the IgE response to ovabumin (OVA) if TCS was given one day before OVA immunization,while OVA alone could not induce IgE to it.In this work,the kinetics of interleukin 4(IL-4) and interferon γ(IFN-γ) gene expression in the mesenteric lymph node (MLN) of TCS-immunized mice was investigated using a semi-quantitative RT-PCR method.It indicated that TCS induced significant IL-4 gene expression and the peaks of IL4 gene expression were on day one after TCS immunization in both primary and secondary response.In contrast,the IFN-γ gene expression was suppressed.Furthermor,the IL-4 gene expression in the secondary response was lower than that in the primary response.Thus the presence of IgE memory B cells were studied.Results showed that the amount of mature IgE mRNA arose significantly and rapidly one day after TCS restimulation,while in the MLN of the mice primed 30 days before and without boost,it was almost as the same amount of the unimmunized control.These findings suggest the existence of the IgE memory B cells in the mice after the primary TCS immunization.     

SDG714 Regulates Specific Gene Expression and Consequently Affects Plant Growth via H3K9 Dimethylation

Histone lysine methylation is known to be involved in the epigenetic regulation of gene expression in all eukaryotes including plants. Here we show that the rice SDG714 is primarily responsible for dimethylation but not trimethylation on histone H3K9 in vivo. Overexpression of YFP-SDG714 in Arabidopsis significantly inhibits plant growth and this inhibition is associated with an enhanced level of H3K9 dimethylation. Our microarray results show that many genes essential for the plant growth and development were downregulated in transgenic Arabidopsis plants overexpressing YFP-SDG714. By chromatin immunoprecipitation analysis, we show that YFP-SDG714 is targeted to specific chromatin regions and dimethylate the H3K9, which is linked with heterochromatinization and the downregulation of genes. Most interestingly, when YFP-SDG714 production is stopped, the inhibited plants can partially restore their growth, suggesting that the perturbation of gene expression caused by YFP-SDG714 is revertible. Taken together, our results point to an important role of SDG714 in H3K9 dimethylation, suppression of gene expression and plant growth, and provide a potential method to regulate gene expression and plant development by an on-off switch of SDG714 expression.     

Flexible manipulation of Omb levels in the endogenous expression region of Drosophila wing by combinational overexpression and suppression strategy

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments.The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms.Driven by a given tissue-specific Gal4,expressing UAS-transgene or UAS-RNAi(RNA interference)could be used to up-or down-regulate target gene expression,respectively.However,the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock.Here,we describe a simple way to modulate optomotor blind(omb)expression levels in its endogenous expression region of the wing disc.We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch.The repression effect is more sensitive to temperature than that of overexpression.At low temperature,overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi.In contrast,at high temperature RNAi predominates in gene expression regulation.By this strategy,we could manipulate omb expression levels at a moderate level.It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.     

Differential gene network analysis from single cell RNA-seq

<正>Study of gene expression has been arguably the most active research field in functional genomics.Over the last two decades,various high-throughput technologies,from gene expression microarray to RNA-seq,have been widely applied to the wholegenome profiling of gene expression.The commonality of these experiments is that they measure the gene expression levels of"bulk"sample,which pools a large number(often in the scale of millions)of cells,and thus the measurements reflect the average expression     

Cloning and Characterization of miRNAs and Their Targets,Including a Novel miRNA-Targeted NBS-LRR Protein Class Gene in Apple （Golden Delicious）

MicroRNA （miRNA） has emerged as an important regulator of gene expression in plants. 146 miRNAs were identified from apple （Malus domestica cv. Golden Delicious） by bioinformatic analysis and RNA library sequencing. From these, 135 were conserved and 11 were novel miRNAs. Target analysis predicted one of the novel miRNAs, Md-miRLn11 （Malus domestica microRNA Ln11）, targeted an apple nucleotide-binding site （NBS）-Ieucine-rich repeat （LRR） class protein coding gene （Md-NBS）. 5/ RACE assay confirmed the ability of Md-miRLn11 to cleave Md-NBS at the 11-12-nt position. Analysis of the expression of Md-miRLn11 and Md-NBS during the optimum invasion period in 40 apple varieties showed that the expression of Md-NBS gene in resistant varieties is higher than in susceptible varieties, with an inverse pattern for Md-miRLn11. Seedlings from the resistant apple variety ＇JiGuan＇ were used to carry out an Agrobacterium infiltration assay, and then inoculated with the apple leaf spot disease. The result showed a clear decline of disease resistance in JiGuan apples. In contrast, the susceptible variety ＇FuJi＇ infiltrated with the Md-NBS gene showed a significant increase in disease resistance. Based on the above results, we propose that Md-miRLn11 regulates Md-NBS gene expression in particular under the condition of pathogen infection, and that the Md-miRLn11 targeting P-loop site may regulate many NBS-LRR protein class genes in woody plants.     

Germline silencing of UASt depends on the piRNA pathway

正The success of the fruit fly Drosophila melanogaster as a model organism is heavily attributed to the expansive range and multitude of genetic and molecular tools available to modify gene expression at will.The Gal4/UAS binary system is one of the most important and widely used genetic tools in Drosophila designed for targeted gene expression(Brand and Perrimon,1993),which allows ectopic expression of any gene(or transgene)in specific tissues,independent of their native regulators.     

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-a) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sp PCC 7120 by triparental conjugative transfer, and the stable transgenic     

Functional properties of a chitinase promoter from cabbage （Brassica oleracea var.capitata）

The 5‘-region of the chitinase gene cabch29,derived from Brassica oleracea var.capitata,has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants.Different 5‘-deletion fragments were linked to reporter gene β-glucuronidase (GUS) as translational fusions,and the expression of these chimeric genes was analyzed in vegetative organs and tissues.Sequences up to-651 showed some basal GUS activity with nearly equal levels in wounded and intact tissues.The addition of further upstream sequences(-651 to-1284) enhanced expression level,and the expression driven by this fragment was inducible by a factor of two to three-fold by wounding.Histochemical analysis of different tissue from transgenic plants that contain cabch29 promoter-gus fusion gene demonstrated woundinducible and tissue-specific cabch29 promoter activity in plants containing the 1308 base pair fragment.The location of GUS activity appears to be cell-specific,being highest in vascular cells and epidermal cells of stem,leaf and roots.Meanwhile,the temporal and spatial expression of cabch29-GUS fusion gene has been investigated.Among the different vegetative organs,a high level of GUS activity was observed in stem and a moderate one in roots;whereas,wounding stress led to a high level of GUS in stem and moderate one in leaf.     

Vascularization and osteogenesis in ectopically implanted bone tissue-engineered constructs with endothelial and osteogenic differentiated adipose-derived stem cells

BACKGROUND A major problem in the healing of bone defects is insufficient or absent blood supply within the defect.To overcome this challenging problem,a plethora of approaches within bone tissue engineering have been developed recently.Bearing in mind that the interplay of various diffusible factors released by endothelial cells(ECs)and osteoblasts(OBs)have a pivotal role in bone growth and regeneration and that adjacent ECs and OBs also communicate directly through gap junctions,we set the focus on the simultaneous application of these cell types together with platelet-rich plasma(PRP)as a growth factor reservoir within ectopic bone tissue engineering constructs.AIM To vascularize and examine osteogenesis in bone tissue engineering constructs enriched with PRP and adipose-derived stem cells(ASCs)induced into ECs and OBs.METHODS ASCs isolated from adipose tissue,induced in vitro into ECs,OBs or just expanded were used for implant construction as followed:BPEO,endothelial and osteogenic differentiated ASCs with PRP and bone mineral matrix;BPUI,uninduced ASCs with PRP and bone mineral matrix;BC(control),only bone mineral matrix.At 1,2,4 and 8 wk after subcutaneous implantation in mice,implants were extracted and endothelial-related and bone-related gene expression were analyzed,while histological analyses were performed after 2 and 8 wk.RESULTS The percentage of vascularization was significantly higher in BC compared to BPUI and BPEO constructs 2 and 8 wk after implantation.BC had the lowest endothelial-related gene expression,weaker osteocalcin immunoexpression and Spp1 expression compared to BPUI and BPEO.Endothelial-related gene expression and osteocalcin immunoexpression were higher in BPUI compared to BC and BPEO.BPEO had a higher percentage of vascularization compared to BPUI and the highest CD31 immunoexpression among examined constructs.Except Vwf,endothelial-related gene expression in BPEO had a later onset and was upregulated and well-balanced during in vivo incubation that induced late onset of Spp1 expression and pronounced osteocalcin immunoexpression at 2 and 8 wk.Tissue regression was noticed in BPEO constructs after 8 wk.CONCLUSION Ectopically implanted BPEO constructs had a favorable impact on vascularization and osteogenesis,but tissue regression imposed the need for discovering a more optimal EC/OB ratio prior to considerations for clinical applications.     

A comparison of honeybee (Apis mellifera) queen,worker and drone larvae by RNA‐Seq

Honeybees (Apis mellifera) have haplodiploid sex determination: males develop from unfertilized eggs and females develop from fertilized ones. The differences in larval food also determine the development of females. Here we compared the total somatic gene expression profiles of 2-day and 4-day-old drone, queen and worker larvae by RNASeq. The results from a co-expression network analysis on all expressed genes showed that 2-day-old drone and worker larvae were closer in gene expression profiles than 2-day-old queen larvae. This indicated that for young larvae (2-day-old) environmental factors such as larval diet have a greater effect on gene expression profiles than ploidy or sex determination. Drones had the most distinct gene expression profiles at the 4-day larval stage, suggesting that haploidy, or sex dramatically affects the gene expression of honeybee larvae. Drone larvae showed fewer differences in gene expression profiles at the 2-day and 4-day time points than the worker and queen larval comparisons (598 against 1190 and 1181), suggesting a different pattern of gene expression regulation during the larval development of haploid males compared to diploid females. This study indicates that early in development the queen caste has the most distinct gene expression profile, perhaps reflecting the very rapid growth and morphological specialization of this caste compared to workers and drones. Later in development the haploid male drones have the most distinct gene expression profile, perhaps reflecting the influence of ploidy or sex determination on gene expression.     

Gene expression profiling in porcine fetal thymus

To obtain an initial overview of gene diversity and expression pattern in porcine thymus, 11,712 ESTs (Expressed Sequence Tags) from 100-day-old porcine thymus (FTY) were sequenced and 7,071 cleaned ESTs were used for gene expression analysis. Clustered by the PHRAP program, 959 contigs and 3,074 singlets were obtained. Blast search showed that 806 contigs and 1,669 singlets (totally 5,442 ESTs) had homologues in GenBank and 1,629 ESTs were novel. According to the Gene Ontology classification, 36.99% ESTs were cataloged into the gene expression group, indicating that although the functional gene (18.78% in defense group) of thymus is expressed in a certain degree, the 100-day-old porcine thymus still exists in a developmental stage. Comparative analysis showed that the gene expression pattern of the 100-day-old porcine thymus is similar to that of the human infant thymus.