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1.
Background Regulatory elements that control expression of specific genes during development have been shown in many cases to contain
functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental
pattern. An example of such a module has been identified at the rat myosin light chain ( MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two
alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development
through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and
the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated
muscle gene expression in the mouse, would drive expression in similar domains in the chicken. 相似文献
3.
Background Bacterial artificial chromosomes (BACs) have been used extensively for sequencing the human and mouse genomes and are thus readily available for most genes. The large size of BACs means that they can generally carry intact genes with all the long range controlling elements that drive full levels of tissue-specific expression. For gene expression studies and gene therapy applications it is useful to be able to retrofit the BACs with selectable genes such as G418 resistance, reporter genes such as luciferase, and oriP/EBNA-1 from Epstein Barr virus which allows long term episomal maintenance in mammalian cells. 相似文献
5.
Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase ( NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants ( NEO+ TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies. 相似文献
6.
Reporter gene imaging is widely used for non-invasively detecting tumorigenesis, trafficking therapeutic cells, and monitoring treatment effect. Baculoviral vectors (BVs) have been utilized as transgenic vectors in the reporter gene imaging systems in recent years. However, BV-mediated report gene imaging can only provide short-term investigation due to its transient transgene expression, which is incompetent for the long-term applications. In the current study, we reconstructed a series of hybrid BVs with several elements, to investigate the feasibility of this hybrid BV-mediated long-term reporter gene imaging in vivo. We showed that with the indispensable assistance of a positive-selection process, hybrid BV containing Sleeping Beauty 100× (SB) transposon system (BV-SB) could significantly prolong the enhanced green fluorescent protein (eGFP) expression for at least 180 days in vitro at nearly 100% eGFP positive percentage and over 1011 arbitrary unit total fluorescence intensity, whereas other hybrid BV-mediated transgene expression gradually faded in only 20 days. Furthermore, BV-SB-mediated eGFP fluorescent reporter gene imaging monitored tumorigenesis in the nude mice for at least 35 days. In addition, we exploited the glucagon-like peptide 1 receptor (glp-1r) gene as a radionuclide reporter gene for in vivo micro-PET imaging. At 50th day post-tumor transplantation, the micro-PET imaging showed considerable radiotracer-receptor-binding in vivo, resulted by stable high level of BV-SB-mediated GLP-1R expression in tumor. In summary, we retrofitted BV with the SB transposon system to make it competent for the long-term reporter gene imaging in vivo, which might broaden the application scopes of BV in the long-term molecular imaging and other biomedicine research fields. 相似文献
7.
Background Transgenic strains of Caenorhabditis elegans are typically generated by injecting DNA into the germline to form multi-copy extrachromosomal arrays. These transgenes are
semi-stable and their expression is silenced in the germline. Mos1 transposon or microparticle bombardment methods have been
developed to create single- or low-copy chromosomal integrated lines. Here we report an alternative method using ultraviolet
trimethylpsoralen (UV/TMP) to generate single/low-copy gene integrations. 相似文献
8.
Background In this study we have built and mined a gene expression database composed of 65 diverse mouse tissues for genes preferentially
expressed in immune tissues and cell types. Using expression pattern criteria, we identified 360 genes with preferential expression
in thymus, spleen, peripheral blood mononuclear cells, lymph nodes (unstimulated or stimulated), or in vitroactivated T-cells. 相似文献
9.
Background Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to
achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug
regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others. 相似文献
11.
Background The Salmonella AvrA gene is present in 80% of Salmonella enterica serovar strains. AvrA protein mimics the activities of some eukaryotic proteins and uses these activities to the pathogen's advantage
by debilitating the target cells, such as intestinal epithelial cells. Therefore, it is important to understand how AvrA works
in targeting eukaryotic signaling pathways in intestinal infection in vivo. In this study, we hypothesized that AvrA interacts with multiple stress pathways in eukaryotic cells to manipulate the host
defense system. A whole genome approach combined with bioinformatics assays was used to investigate the in vivo genetic responses of the mouse colon to Salmonella with or without AvrA protein expression in the early stage (8 hours) and late stage (4 days). Specifically, we examined the
gene expression profiles in mouse colon as it responded to pathogenic Salmonella stain SL1344 (with AvrA expression) or SB1117 (without AvrA expression). 相似文献
12.
The Sleeping Beauty (SB) transposase is the most active transposase in vertebrate cells, and the SB transposon system has been used as a tool for insertional mutagenesis and gene delivery. Previous studies have indicated that the frequency of chromosomal transposition is considerably higher in mouse germ cells than in mouse embryonic stem cells, suggesting the existence of unknown mechanisms that regulate SB transposition. Here, we demonstrated that CpG methylation of the transposon region enhances SB transposition. The transposition efficiencies of a methylated transposon and an unmethylated transposon which had been targeted in the same genomic loci by recombination-mediated cassette exchange in mouse erythroleukemia cells were compared, and at least a 100-fold increase was observed in the methylated transposon. CpG methylation also enhanced transposition from plasmids into the genome. Chromatin immunoprecipitation assays revealed that histone H3 methylated at lysine-9, a hallmark of condensed heterochromatin, was enriched at the methylated transposon, whereas the unmethylated transposon formed a relaxed euchromatin structure, as evidenced by enrichment of acetylated histone H3 and reporter gene expression. Possible roles of heterochromatin formation in the transposition reaction are discussed. Our findings indicate a novel relationship between CpG methylation and transposon mobilization. 相似文献
13.
Background Gene promoters fused to the firefly luciferase gene ( luc) are useful for examining gene regulation in live transgenic mice and they provide unique views of functioning organs. The
dynamics of gene expression in cells and tissues expressing luciferase can be observed by imaging this enzyme's bioluminescent
oxidation of luciferin. Neural pathways involved in specific behaviors have been identified by localizing expression of immediate-early
genes such as c- fos. A transgenic mouse line with luc controlled by the human c- fos promoter (fos::luc) has enabled gene expression imaging in brain slice cultures. To optimize imaging of immediate-early gene
expression throughout intact mice, the present study examined fos::luc mice and a second transgenic mouse containing luc controlled by the human cytomegalovirus immediate-early gene 1 promoter and enhancer (CMV::luc). Because skin pigments and
hair can significantly scatter light from underlying structures, the two transgenic lines were crossed with a hairless albino
mouse (HRS/J) to explore which deep structures could be imaged. Furthermore, live anesthetized mice were compared with overdosed
mice. 相似文献
15.
Background The complexity of the mouse mu opioid receptor ( Oprm) gene was demonstrated by the identification of multiple alternatively spliced variants and promoters. Our previous studies
have identified a novel promoter, exon 11 (E11) promoter, in the mouse Oprm gene. The E11 promoter is located ~10 kb upstream of the exon 1 (E1) promoter. The E11 promoter controls the expression of
nine splice variants in the mouse Oprm gene. Distinguished from the TATA-less E1 promoter, the E11 promoter resembles a typical TATA-containing eukaryote class
II promoter. The aim of this study is to further characterize the E11 and E1 promoters in vivo using a transgenic mouse model. 相似文献
16.
Background Despite of the fact that mammalian genomes are far more spacious than prokaryotic genomes, recent nucleotide sequencing data
have revealed that many mammalian genes are arranged in a head-to-head orientation and separated by a small intergenic sequence.
Extensive studies on some of these neighboring genes, in particular homologous gene pairs, have shown that these genes are
often co-expressed in a symmetric manner and regulated by a shared promoter region. Here we report the identification of two
non-homologous brain disease-related genes, with one coding for a serine protease inhibitor ( SERPINI1) and the other for a programmed cell death-related gene ( PDCD10), being tightly linked together by an asymmetric bidirectional promoter in an evolutionarily conserved fashion. This asymmetric
bidirectional promoter, in cooperation with some cis-acting elements, is responsible for the co-regulation of the gene expression
pattern as well as the tissue specificity of SERPINI1 and PDCD10. 相似文献
18.
Background Recent circadian clock studies using gene expression microarray in two different tissues of mouse have revealed not all circadian-related
genes are synchronized in phase or peak expression times across tissues in vivo. Instead, some circadian-related genes may be delayed by 4–8 hrs in peak expression in one tissue relative to the other.
These interesting biological observations prompt a statistical question regarding how to distinguish the synchronized genes
from genes that are systematically lagged in phase/peak expression time across two tissues. 相似文献
19.
Background DNA transposons have emerged as indispensible tools for manipulating vertebrate genomes with applications ranging from insertional
mutagenesis and transgenesis to gene therapy. To fully explore the potential of two highly active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have conducted a side-by-side comparison of the two transposon systems in the same setting
to evaluate their advantages and disadvantages for use in gene therapy and gene discovery. 相似文献
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