As an effective vehicle for bio-research and for gene therapy, Lentiviral Vector (LV) has been drawn large attention in recent years. However, transcriptional read-through limits its application. In order to understand the extend of LV read-through in chromosome, a reliable method to assess transcriptional read-through rate is needed. Here, we report the method as follows: 293T cells were transfected with the lentiviral transfer vectors which borne with two LTRs at its two ends in order to mimic the state of "proviral vectors" in chromosome. Using the primers specific for 3'U5 and 3'U3, read-through and total transcripts were reverse transcribed, respectively. These two cDNAs were quantified by realtime PCR using the primers and probe specific for 5'end of 3'U3. Read-through rate was then calculated by the division of the two. Meanwhile, read-through product of green fluorescence protein was also analyzed by Fluorescence Activated Cell Sorter. They both reciprocally proved the principal and confirmed that self-inactivated LV appeared higher read-through rate than the wild type one. The method described in this article, therefore, provides a useful technique to study how to reduce read-through rate, and improve the bio-safety of LV.
Flow Cytometry ; Genetic Therapy ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; HEK293 Cells ; Humans ; Lentivirus ; Transfection

Fusobacterium nucleatum (F. nucleatum) is an early pathogenic colonizer in periodontitis, but the host response to infection with this pathogen remains unclear. In this study, we built an F. nucleatum infectious model with human periodontal ligament stem cells (PDLSCs) and showed that F. nucleatum could inhibit proliferation, and facilitate apoptosis, ferroptosis, and inflammatory cytokine production in a dose-dependent manner. The F. nucleatum adhesin FadA acted as a proinflammatory virulence factor and increased the expression of interleukin(IL)-1β, IL-6 and IL-8. Further study showed that FadA could bind with PEBP1 to activate the Raf1-MAPK and IKK-NF-κB signaling pathways. Time-course RNA-sequencing analyses showed the cascade of gene activation process in PDLSCs with increasing durations of F. nucleatum infection. NFκB1 and NFκB2 upregulated after 3 h of F. nucleatum-infection, and the inflammatory-related genes in the NF-κB signaling pathway were serially elevated with time. Using computational drug repositioning analysis, we predicted and validated that two potential drugs (piperlongumine and fisetin) could attenuate the negative effects of F. nucleatum-infection. Collectively, this study unveils the potential pathogenic mechanisms of F. nucleatum and the host inflammatory response at the early stage of F. nucleatum infection.
Humans ; Fusobacterium nucleatum/metabolism* ; NF-kappa B/metabolism* ; Periodontal Ligament/metabolism* ; Signal Transduction ; Fusobacterium Infections/pathology* ; Stem Cells/metabolism*