This study was aimed to establish a high-throughput luciferase reporter system, through which to screen and identify miRNAs directly targeting p21, and to explore the biological function and significance of these miRNAs. Molecular cloning technique was used to construct and identify two lentivirus-expressing vectors-pWPXL-Luc and pWPXL-Luc-P21-3'UTR, virus particles were collected after the pWPXL-Luc or pWPXL-Luc-P21-3'UTR vectors were co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pDM2G into HEK-293T cells. Furthermore, two stable cell lines expressing luciferase singly or co-expressed luciferase and P21-3'UTR were established by transducing HEK-293 cells with recombinant lentivirus; the former was used as control in the following experiments. Finally luciferase activity of the latter stable cells was measured by using the luciferase reporter assay system. The results showed that high-titre recombinant lentivirus was produced and two stable cell lines were constructed, also to some certain, the luciferase activity was in direct proportion to the number of cells. In conclusion, the high-throughput luciferase reporter system is established successfully; using this system, the 28 miRNA that directly target P21 Cip1/Waf1 are screened experimentally.
Genes, Reporter ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; genetics ; Luciferases ; genetics ; MicroRNAs ; genetics ; Plasmids ; Transduction, Genetic

In this study, the recombinant adenovirus (Ad) vector containing dual reporter gene [i.e. human transferrin receptor gene (TFRC) and firefly luciferase reporter gene] was constructed to provide a novel experimental tool for magnetic resonance (MR) and bioluminescence dual-modality molecular imaging. The cDNA of TFRC was amplified by polymerase chain reaction (PCR) and cloned into the multiple cloning site of pShuttle-CMV-CMV-Luciferase vector. After identification by Sfi I digestion and sequencing, pShuttle-TFRC-Luciferase vector and the adenoviral backbone vector (pAdeno) were subjected to homologous recombination. The correct recombinant plasmid was then transfected into 293 packaging cells to produce adenoviral particles and confirmed by PCR. After infection of human colorectal cancer LOVO cells with Ad-TFRC-Luciferase, the expressions of transferrin receptor (TfR) and luciferase protein were detected respectively by Western blotting and bioluminescence imaging in vitro. The results showed that TFRC gene was successfully inserted into the adenoviral shuttle vector carrying luciferase gene. DNA sequence analysis indicated that the TFRC gene sequence in the shuttle plasmid was exactly the same as that reported in GenBank. The recombinant plasmid was identified correct by restriction digestion. Ad-TFRC-Luciferase recombinant adenovirus was constructed successfully, and the virus titer was 1.6×10(10) pfu/mL. Forty-eight h after dual reporter gene transfection, the expressions of TfR and luciferase protein were increased significantly (P<0.01). It was concluded that the recombinant adenovirus vector with dual reporter gene was successfully established, which may be used for in vivo tracing target cells in multimodality imaging.
Adenoviridae ; genetics ; Genes, Reporter ; genetics ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; Molecular Imaging ; methods

Vitamin E (Tocopherols) is lipid-soluble antioxidants and essential for human health. Gamma-tocopherol methyltransferase (delta-TMT), one of the key enzymes in tocopherol biosynthetic pathway in plants, converts delta,sigma-tocopherols into alpha,beta-tocopherols. In this study, we isolated the 1552 bp promoter of Arabidopsis TMT gene. The promoter was fused with GUS reporter gene and this expression cassette was introduced into wild Arabidopsis thaliana by Agrobacterium-mediated transformation. GUS staining shows that GUS gene is expressed in leaves, stems and flowers, with the highest expression in young leaves, stamens and stem apices, while not observable in roots, seeds and siliques. The data indicate that gamma-TMT gene promoter is likely to be expressed preferentially in some of the tissues of Arabidopsis.
Arabidopsis ; genetics ; Base Sequence ; Genes, Reporter ; Immunohistochemistry ; Methyltransferases ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics

Genes, Reporter ; Genetic Vectors ; Luciferases ; genetics ; Transcription, Genetic ; bcl-2-Associated X Protein ; genetics

The transposon vector containing enhanced green fluorescent protein (EGFP) was injected into early housefly (Musca domestica L.) eggs by microinjection method to realize stable gene expression in vivo for verification, and to study housefly gene function. A borosilicate glass micro injection needle suitable for microinjection of housefly eggs was made, the softening treatment conditions of housefly egg shells were explored, and a microinjection technology platform suitable for housefly was constructed with a high-precision microsyringe Nanoject Ⅲ as the main body. The recombinant plasmid PiggyBac-[3×P3]-EGFP containing the eye-specific 3×P3 promoter and EGFP and the stable genetic expression helper plasmid pHA3pig helper were microinjected into the treated housefly eggs. After emergence, the eye luminescence was observed, and the expression and transcription level of EGFP were detected. The results showed that the normal hatching rate of housefly eggs was 55% when rinsed in bleaching water for 35 s. The hardness of the egg shell treated for 35 s was suitable for injection and the injection needle was not easy to break. About 3% of the emerged housefly eyes had green fluorescence. Through further molecular detection, EGFP specific fragments with a size of 750 bp were amplified from DNA and RNA of housefly. Through the technical platform, the stable expression of reporter genes in housefly can be conveniently and effectively realized, and a bioreactor with housefly as the main body can be established, which provides certain reference value for subsequent research on housefly gene function.
Animals ; Animals, Genetically Modified ; Gene Expression ; Genes, Reporter ; Green Fluorescent Proteins/genetics* ; Houseflies/genetics* ; Microinjections

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.
Cloning, Molecular ; Escherichia coli/genetics* ; Genes, Reporter/genetics* ; Genetic Vectors/genetics* ; Lac Operon/genetics* ; Plasmids/genetics* ; beta-Galactosidase/genetics*

OBJECTIVETo investigate the know gene types of main wild type measles virus strains and take measures to control measles in Jilin Province.

METHODSGenetic characterization of 9 measles viruses isolated from 72 throat swabs or urine specimens of measles patients using CDW(150) cells line was studied in Jilin Province in 2005.

RESULTSSequence analysis of 450 nucleotides of COOH-terminal of nucleoprotein (N) genes of 9 isolates indicated that all were members of H(1) genotype, in which there are 7 strains of H1a and 2 strains of H1b, the H1a subgroup differed from H1b by 2.0% approximately 3.5% at the nucleotide level in the COOH-terminal of the N gene.

CONCLUSIONSThe H(1) genotype of wild-type measles viruses should be the main epidemic strain and main pathogen that caused measles outbreaks and sporadic cases in Jilin Province.

China ; Genes, Reporter ; Genes, Viral ; Genotype ; Humans ; Measles ; epidemiology ; virology ; Measles virus ; genetics ; Molecular Sequence Data ; Viral Structural Proteins ; genetics

OBJECTIVETo develop an alternative method for assessment of gene delivery systems in vivo.

METHODSMouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase (Gluc) expression cassette. After implantation of these cells into recipient mice, the expression of Gluc was detected in whole blood or plasma collected.

RESULTSAs little as 10 muL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer. And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice.

CONCLUSIONSGluc may be useful as an in vivo reporter for gene therapy researches, and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.

Animals ; Arecaceae ; enzymology ; Cell Line ; Gene Transfer Techniques ; Genes, Reporter ; Humans ; Luciferases ; genetics ; Mice

Chromosomal integration of heterologous genes or pathways is preferred over the use of episomal plasmids for its inherently stability and thus more desirable in the industrial setting. However, the position of integration of heterologous genes in the genome influences the expression levels. In combination of high throughput transformation of the Yeast Knock-out Collection (YKO) and FACS analysis, the position effect on heterologous reporter gene gfp was identified across the whole genome in yeast. In total 428 high-expressed sites and 444 low-expressed sites were spotted, providing massive data to analyze patterns and reasons for region dependency of gene expression on the genome-wide scale.
Gene Expression Regulation, Fungal ; Gene Knock-In Techniques ; Genes, Reporter ; Genome, Fungal ; Saccharomyces cerevisiae ; genetics

This study was aimed to obtain higher efficiency in gene transfection into K562 cells and to study the role of green fluorescence protein (GFP) as a reporter system. Transfection efficiencies with different methods including electroporation and lipofectamine 2000 transfection, were observed and calculated under fluorescent microscopy by using GFP as a reporter system. The results showed that the transfection efficiency with electroporation (10%) was higher than that with lipofectamine 2000 (1%). In conclusion, the electroporation is a more ideal method for introduction of foreign gene into K562 cells. GFP can be used as a reporter system for optimizing transfection of K562 cells.
Electroporation ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; Humans ; K562 Cells ; Liposomes ; Transfection