Recombinant Interferon-γ Lentivirus Co-Infection Inhibits Adenovirus Replication Ex Vivo

Zhang, Ling; Yin, Sha; Tan, Wanlong; Xiao, Dong; Weng, Yunceng; Wang, Wenjing; Li, Tingting; Shi, Junpeng; Shuai, Lifang; Li, Hongwei; Zhou, Jinlin; Allain, Jean‐Pierre

doi:10.1371/journal.pone.0042455

Cited by 6 publications

(8 citation statements)

References 32 publications

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“…The lentivirus carrying shRNA against ZFP36L2, clone TRCN0000175839, with targeting sequence 5Ј-TTTCA TATTGAGTTGTGGTGC-3Ј showed the highest knockdown of ZFP36L2. The effectiveness of viral transduction was monitored by qPCR against lentiviral DNA relative to mouse genomic DNA obtained from the infected DRG, using primers in viral 3Ј-LTR LV forward 5Ј-TAAAGCTTGCCTTGAGTGCT-3Ј, LV-R 5Ј-GTCTGAGGGATCTCTA GTTACCAG-3Ј, as described previously (Zhang et al, 2012). A scrambled shRNA in pLKO.1 vector (shControl-2) was obtained from Addgene (plasmid #1864) with targeting sequence of 5Ј-CCTAAGGTT AAGTCGCCCTCGCTC-3Ј.…”

Section: Methodsmentioning

confidence: 99%

An RNA Binding Protein Promotes Axonal Integrity in Peripheral Neurons by Destabilizing REST

et al. 2014

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The RE1 Silencing Transcription Factor (REST) acts as a governor of the mature neuronal phenotype by repressing a large consortium of neuronal genes in non-neuronal cells. In the developing nervous system, REST is present in progenitors and downregulated at terminal differentiation to promote acquisition of mature neuronal phenotypes. Paradoxically, REST is still detected in some regions of the adult nervous system, but how REST levels are regulated, and whether REST can still repress neuronal genes, is not known. Here, we report that homeostatic levels of REST are maintained in mature peripheral neurons by a constitutive post-transcriptional mechanism. Specifically, using a three-hybrid genetic screen, we identify the RNA binding protein, ZFP36L2, associated previously only with female fertility and hematopoiesis, and show that it regulates REST mRNA stability. Dorsal root ganglia in Zfp36l2 knock-out mice, or wild-type ganglia expressing ZFP36L2 shRNA, show higher steady-state levels of Rest mRNA and protein, and extend thin and disintegrating axons. This phenotype is due, at least in part, to abnormally elevated REST levels in the ganglia because the axonal phenotype is attenuated by acute knockdown of REST in Zfp36l2 KO DRG explants. The higher REST levels result in lower levels of target genes, indicating that REST can still fine-tune gene expression through repression. Thus, REST levels are titrated in mature peripheral neurons, in part through a ZFP36L2-mediated post-transcriptional mechanism, with consequences for axonal integrity.

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Section: Methodsmentioning

confidence: 99%

An RNA Binding Protein Promotes Axonal Integrity in Peripheral Neurons by Destabilizing REST

et al. 2014

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“…The CMV promoter (with or without β-globin intron) resulted in the highest initial level of GFP expression amongst the vectors, indicating its ability for rapid and high-level transgenic expression in 293T cell line. This finding pinpoints to the importance of a proper choice of promoter in achieving high yield of transgene production in recombinant cell lines 19 .…”

Section: Discussionmentioning

confidence: 93%

Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

Mao

Yan

et al. 2015

Int. J. Med. Sci.

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Objectives:Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors.Methods: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p'HR.cppt.3'1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution.Results:GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (β-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction. Surprisingly, neither the HS4 insulator nor the UCOE sequence improved the GFP expression level or stability. Clonal cell lines with HCV E1 gene were generated from LV-CMV-E1 vector-infected 293T cells. A representative recombinant cell line maintained stable E1expression for at least 9 weeks without significant difference in morphology compared with untreated 293T cells.Conclusion: The results suggest that all five vectors can stably transduce 293T cells, producing long term transgene expression with different efficiencies. However, neither the insulator nor the UCOE improved the GFP expression. The vectors containing the promoter CMV or CMV (β-globin intron) generated the highest gene expressions, manifesting as more favorable candidates for recombinant protein production in HEK293T cells.

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“…Omp31 was expressed in 293FT cells by Omp31-lentivirus-mediated transduction as described previously [30], mimicking Omp31 antigen in Brucella -infected mammalian cells.…”

Section: Methodsmentioning

confidence: 99%

Characterization of novel Omp31 antigenic epitopes of Brucella melitensis by monoclonal antibodies

Chen

et al. 2017

BMC Microbiol

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BackgroundBrucellosis is a severe zoonotic disease worldwide. Detection and identification of Brucella species are essential to prevent or treat brucellosis in humans and animals. The outer membrane protein-31 (Omp31) is a major protein of Brucellae except for B. abortus, while the Omp31 antigenic epitopes have not been extensively characterized yet. ResultsA total of 22 monoclonal antibodies (mAbs) were produced against Omp31 of Brucella (B.) melitensis, of which 13 recognized five linear epitopes, 7 reacted with semi-conformational epitopes and 2 reacted with conformational epitopes, respectively. The mAb isotypes were 11 (50%) IgG2a, 5 (23%) IgG1 and 6 (27%) IgM. On the basis of epitope recognition and reactivity levels, 8 mAbs including 3 IgM and 5 IgG clones were considered as highly reactive and potentially diagnostic antibodies. Among these mAbs, 7A3 (IgG1), 5B1 (IgG2a), 2C1 (IgG2a) and 5B3 (IgG2a) reacted with differently conserved linear epitopes of B. melitensis, B. ovis, B. suis and B. canis strains, while 5H3 (IgG2a) highly reacted with a conformational epitope of Omp31 when tested with several immunoassays.ConclusionsThese potent monoclonal antibodies can be used for identifying Omp31 antigens or detecting B. melitensis and other Brucella species beyond B. abortus in vitro or in vivo.

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Recombinant Interferon-γ Lentivirus Co-Infection Inhibits Adenovirus Replication Ex Vivo

Cited by 6 publications

References 32 publications

An RNA Binding Protein Promotes Axonal Integrity in Peripheral Neurons by Destabilizing REST

An RNA Binding Protein Promotes Axonal Integrity in Peripheral Neurons by Destabilizing REST

Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

Characterization of novel Omp31 antigenic epitopes of Brucella melitensis by monoclonal antibodies

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