Immunotherapy is becoming an effective and less invasive strategy that can be applied to the treatment of various malignancies. Lentiviral vectors (LVs) have shown great potential in immunotherapy as they can stably integrate relatively large foreign DNA, and effectively transduce dividing and non-dividing cells. Clinical application needs high quality LVs, and therefore strict quality control of the final products is necessary to ensure their purity, efficacy and safety. The quantitative detection of LVs is among the key parts of product development and quality control. In this paper, the existing methods for quantitative detection of LVs are summarized, including fluorescence activated cell sorter (FACS), P24 enzyme-linked immuno sorbent assay (P24 ELISA), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing(TRPS) and virus counter(VC).Their advantages and disadvantages are listed, and future development and challenges are discussed.
Genetic Vectors/genetics* ; Humans ; Immunotherapy ; Lentivirus/genetics* ; Neoplasms ; Transduction, Genetic

Viruses are powerful tools for the study of modern neurosciences. Most of the research on the connection and function of neurons were done by using recombinant viruses, among which neurotropic herpesvirus is one of the most important tools. With the continuous development of genetic engineering and molecular biology techniques, several recombinant neurophilic herpesviruses have been engineered into different viral tools for neuroscience research. This review describes and discusses several common and widely used neurophilic herpesviruses as nerve conduction tracers, viral vectors for neurological diseases, and lytic viruses for neuro-oncology applications, which provides a reference for further exploring the function of neurophilic herpesviruses.
Herpesviridae/genetics* ; Neurosciences ; Genetic Vectors/genetics* ; Genetic Engineering ; Neurons

Dependovirus ; genetics ; Genetic Therapy ; Genetic Vectors ; Humans ; Liver Diseases ; therapy

The development of the genetic transformation systems in extremely halophilic Archaea was reviewed in this paper. Included are the screening of selectable markers for resistance to antibiotics, the development of gene cloning and expression vectors, and the modifications of the host organisms.
Archaea ; genetics ; Cloning, Molecular ; Genetic Vectors ; Transformation, Genetic

Gene therapy has been considered as one of the optimal treatments. Although, at the beginning of this century, a series of unexpected side effects brought gene therapy into depression, the improved lentiviral vectors, which characterised by high efficiency transfection, stable expression in target cells and good biosafety, have been applied in clinical trials in recent years and acquired a certain clinical improvements. Nowadays gene therapy becomes an eye-catching field. This review discusses the gene therapy how blocked by lentiviral vectors, the high efficiency and biosafety of lentiviral vectors, the improvement of lentiviral vector preparation and so on.
Animals ; Genetic Therapy ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Transfection

The technology of genetic engineering has been widely used to express macromolecules such as enzymes. However, it is difficult to detect and purify the micromolecules such as small peptides, because of their instability and degradability. Construction of multi-copy recombinant expression plasmid can be achieved by inserting multiple target genes or expression cassette containing target genes with the same orientation into expression vector. This is effective to increase the expression level of small peptides. In this article we described four methods in order to provide some optional methods and ideas for the expression of active small peptides.
Gene Expression ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; metabolism ; Peptides ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

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

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

A safe and effective targeting viral vector is the key factor for successful clinical gene therapy. microRNA, a class of small, single-stranded endogenous RNAs, act as post-transcriptional regulators of gene expression. The discovery of these kind regulatory elements provides a new approach to regulate gene expression more accurately. In this review, we elucidated the principle of microRNA in regulation of targeting viral vector. The applications of microRNA in the fields of elimination contamination from replication competent virus, reduction of transgene-specific immunity, promotion of cancer-targeted gene therapy and development of live attenuated vaccines were also discussed.
Gene Expression Regulation ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; MicroRNAs ; genetics ; Viruses ; genetics ; metabolism

Manipulation of genes, including knock-out or knock-in, replacement of gene elements (such as promoters), fusion with a fluorescent protein gene, and construction of in situ gene reporter, is required in most of the biotechnological laboratories. The widely used gene manipulating methods based on two-step allelic exchange are cumbersome in terms of constructing plasmids, transforming and screening. In addition, the efficiency of using this method for long fragment knockout is low. To simplify the process of gene manipulation, we constructed a minimized integrative vector pln2. When a gene needs to be inactivated, an internal fragment of the target gene (non-frameshift) is cloned into the pln2 plasmid. Once the single-crossover recombination between genome and the constructed plasmid occurs, the endogenous gene is segmented by the plasmid backbone and thus inactivated. We developed a toolbox based on pln2 that can be used for different genomic operation mentioned above. With the help of this toolbox, we successfully knocked out large fragments of 20-270 kb.
Genetic Vectors/genetics* ; Pseudomonas aeruginosa/genetics* ; Plasmids/genetics* ; Promoter Regions, Genetic ; Genome