Dependovirus/genetics* ; Microglia ; Cells, Cultured ; Transduction, Genetic

Gene therapy is a rapidly developing field. The most widely used technique for foreign gene transfer is lentiviral-mediated gene therapy. Lentiviral vector has been developed from the first generation to the third generation in terms of safety. The preparation of lentiviruses with high titer remains difficult. In this study, a Fibra-Cel sheet carrier was used as an HEK293T cell carrier matrix, and several sterile cell culture spinners were combined and cultured on a roller bottle machine to scale up the adherent cells. The virus titer was maximized by screening the factors to optimize the lentivirus titer in the third-generation lentivirus packaging process one by one. Fibra-Cel sheet vector was successfully used as the matrix of HEK293T cell adhesion to culture adherent cells at large scale. The optimal conditions for large-scale preparation of the third-generation lentivirus by bottle roller were screened and three batches of lentiviruses were produced on pilot scale. The production time of lentivirus was shortened from 120 hours to 54 hours from plasmid transfection to virus collection; in terms of cost, a rolling bottle machine was used instead of a bioreactor, leading to lower cost and no need for repeated sterilization during the whole process. The safe, effective and low-cost operation of successful production will provide a technical base for the large-scale preparation of lentivirus and thus lay a firm foundation for its clinical application.
Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; Transduction, Genetic ; Transfection

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

Cationic liposome is a greatly promising gene carrier. In this paper, the structure trait of cationic liposome was briefly introduced, the mechanism of cationic liposomes mediated gene delivery and the main influencing factor of transfection efficiency in the transfection process were discussed chiefly.
Cations ; chemistry ; Genetic Therapy ; Genetic Vectors ; genetics ; Liposomes ; chemistry ; Transduction, Genetic ; Transfection

Functional analysis of new genes is playing a central role in postgenomic era. Here we reviewed several main strategies including bioinformatics, gene transduction, antisense technology, certain gene silence induced by RNA interference (RNAi), transgene and gene knockout and artificial chromosome transduction.
Animals ; Computational Biology ; methods ; Genes ; physiology ; Humans ; Transduction, Genetic ; methods

Recombinant adeno-associated virus (rAAV) has been widely used as vector for gene therapy. However, the effectiveness of gene therapy based on rAAV needs to be further improved. Enhancement of the transduction efficiency is one of the most important fields for rAAV-based gene therapy. Recent results have showed that the ubiquitin-proteasome system plays an important role in the trafficking of rAAV vector in cytoplasm, and regulation of its function may significantly improve the transduction efficiency of rAAV vector in various types of cells and tissues.
Animals ; Dependovirus ; genetics ; metabolism ; Genetic Vectors ; genetics ; metabolism ; Humans ; Transduction, Genetic ; Ubiquitin ; metabolism

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

OBJECTIVE: To investigate the expression efficiency of exogenous gene mediated by different serotypes of adeno-associated virus (AAV) vectors in retina, and to compare the expression efficiency of AAV vector and two kinds of promoters commonly used in ophthalmology after transfection into mouse retina, so as to provide the basis for selecting appropriate AAV vector and promoter for gene therapy of retinitis pigmentosa. METHODS: AAV2/2, AAV2/5, AAV2/8 and AAV2/9 were prepared. The C57BL/6J mice were injected subretinally with 1 μL purified AAV vectors (1.00×10¹³ mg/L). Then the mice were killed 2 or 4 weeks after treatment, and the eyes were enucleated for frozen section. The expression of green fluorescent protein (GFP) was observed under the confocal microscope. Two kinds of promoters, CMV and CAG, were selectd, and the expression of AAV2/8-GFP-CMV and AAV2/8-GFP-CAG was observed under confocal microscope. RESULTS: No bacterial infection or immune response were seen in the injected mice. 2 weeks after injection, the GFP green fluorescence of AAV2/8 and AAV2/9 in the mouse retina was obvious, which indicated that the GFP green fluorescence of AAV2/8 and AAV2/9 was high after transfection into the mouse retina. In these two serotypes, GFP green fluorescence of AAV2/8 was mainly concentrated in photoreceptor cells while AAV2/8 was expressed in the whole retina, indicating that AAV2/8 was more specific to photoreceptors. Further experiments on AAV2/8 showed that the GFP green fluorescence of the mouse retina was obvious 4 weeks after injection, indicating that the exogenous gene mediated by AAV2/8 could be stably expressed in vivo. For CMV and CAG promoters, CMV promoter was expressed stronger in retinal pigment epithelium (RPE)cells, while CAG promoter was stronger in photorecepters. In photorecepters, CAG promoter was expressed almost the same as CMV promoter, while CMV promoter was stronger in RPE cells. CONCLUSION AAV vectors could express transgene robustly in retinal cells; Among several AAV serotypes, AAV2/2 and AAV2/5 showed weaker GFP fluorescence than AAV2/8 and AAV2/9. AAV2/9 showed expression in each layer of the retina including ganglion cells. AAV2/8 was more specific for photoreceptor; CAG promoters had higher specificity for photoreceptors than CMV promoters.
Animals ; Dependovirus/genetics* ; Genetic Vectors ; Mice ; Mice, Inbred C57BL ; Retina ; Serogroup ; Transduction, Genetic

OBJECTIVE: To screen better promoters and provide more powerful tools for basic research and gene therapy of hemophilia. METHODS: Bioinformatics methods were used to analyze the promoters expressing housekeeping genes with high abundance, so as to select potential candidate promoters. The GFP reporter gene vector was constructed, and the packaging efficiency of the novel promoter was investigated with EF1 α promoter as control, and the transcription and activities of the reporter gene were investigated too. The activity of the candidate promoter was investigated by loading F9 gene. RESULTS: The most potential RPS6 promoter was obtained by screening. There was no difference in lentiviral packaging between EF1 α-LV and RPS6-LV, and their virus titer were consistent. In 293T cells, the transduction efficiency and mean fluorescence intensity of RPS6pro-LV and EF1 αpro-LV were proportional to the lentiviral dose. The transfection efficiency of both promoters in different types of cells was in the following order: 293T>HEL>MSC; Compared with EF1 αpro-LV, RPS6pro-LV could obtain a higher fluorescence intensity in MSC cells, and RPS6pro-LV was more stable in long-term cultured HEL cells infected with two lentiviruses respectively. The results of RT-qPCR, Western blot and FIX activity (FIX∶C) detection of K562 cell culture supernatant showed that FIX expression in the EF1 α-F9 and RPS6-F9 groups was higher than that in the unloaded control group, and there was no significant difference in FIX expression between the EF1 α-F9 and RPS6-F9 groups. CONCLUSION After screening and optimization, a promoter was obtained, which can be widely used for exogenous gene expression. The high stability and viability of the promoter were confirmed by long-term culture and active gene expression, which providing a powerful tool for basic research and clinical gene therapy of hemophilia.
Humans ; Transduction, Genetic ; Genetic Vectors ; Hemophilia A/genetics* ; Transfection ; Blood Coagulation Factors/genetics* ; Lentivirus/genetics*

There are many E. coli rare codons in the gene of porcine interferon alpha-1. In order to obtain high expression of poIFN-alpha1 in E. coli, the cDNA encoded poIFN-alpha1 mature protein was synthesized using biased codons of E. coli without changing the original amino acid sequence and the terminator was changed as TAA. At the same time, Adenine and Thymine were used to the largest extent near the 5' terminus of poIFN-alpha1 mature protein gene. The synthesized gene was inserted into the Eco RI and Sal I site of the expression vector pRLC resulting pRLC-poIFN-alpha1. The poIFN-alpha1 is highly expressed in E. coli DH5alpha when the induction was carried out at 42 degrees C . The expressed poIFN-alpha1 account for 24.5% of the total cellular proteins and existed as inclusion body. The poIFN-alpha1 inclusion body was dissolved in 6mol/L guanidine chloride contained DTT and subsequently the denatured poIFN-alpha1 was re-natured by dilution in refolding buffer containing GSH and GSSH. In the present study it was found that the denatured poIFN-alpha1 was most efficiently re-natured in refolding buffer containing 1 mol/L guanidine chloride. In order to obtain pure protein, the concentrated re-natured poIFN-alpha1 was purified by Sephacryl S-200 chromatography. As a result, the purified poIFN-alpha1 is verified to be of high cytokine activity by inhibiting the cytopathic effect of vesicular stomatitis virus in MDBK cells, which is about 6.4 x 10(6) u/mg. This study paved the way for large-scale production of recombinant poIFN-alpha1 and its usage in virus disease control of pigs.
Animals ; Codon ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Interferon-alpha ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Swine ; Transduction, Genetic