Development of a Lentivirus Vector-Based Assay for Non-Destructive Monitoring of Cell Fusion Activity

Abstract

Cell-to-cell fusion can be quantified by endowing acceptor and donor cells with latent reporter genes/proteins and activators of these genes/proteins, respectively. One way to accomplish this goal is by using a bipartite lentivirus vector (LV)-based cell fusion assay system in which the cellular fusion partners are transduced with a flippase-activatable Photinus pyralis luciferase (PpLuc) expression unit (acceptor cells) or with a recombinant gene encoding FLPeNLS+, a nuclear-targeted and molecularly evolved version of flippase (donor cells). Fusion of both cell populations will lead to the FLPe-dependent generation of a functional PpLuc gene. PpLuc activity is typically measured in cell lysates, precluding consecutive analysis of one cell culture. Therefore, in this study the PpLuc-coding sequence was replaced by that of Gaussia princeps luciferase (GpLuc), a secretory protein allowing repeated analysis of the same cell culture. In myotubes the spread of FLPeNLS+ may be limited due to its nuclear localization signal (NLS) causing low signal outputs. To test this hypothesis, myoblasts were transduced with LVs encoding either FLPeNLS+ or an NLS-less version of FLPe (FLPeNLS−) and subsequently co-cultured in different ratios with myoblasts containing the FLPe-activatable GpLuc expression cassette. At different times after induction of cell-to-cell fusion the GpLuc activity in the culture medium was determined. FLPeNLS+ and FLPeNLS− both activated the latent GpLuc gene but when the percentage of FLPe-expressing myoblasts was limiting, FLPeNLS+ generally yielded slightly higher signals than FLPeNLS− while at low acceptor-to-donor cell ratios FLPeNLS− was usually superior. The ability of FLPeNLS+ to spread through myofibers and to induce reporter gene expression is thus not limited by its NLS. However, at high FLPe concentrations the presence of the NLS negatively affected reporter gene expression. In summary, a rapid and simple chemiluminescence assay for quantifying cell-to-cell fusion progression based on GpLuc has been developed.

SLC6A8 Knockdown Suppresses the Invasion and Migration of Human Hepatocellular Carcinoma Huh-7 and Hep3B Cells

“… 34 , 35 On the other hand, the role of SLC6A8 in human hepatocellular carcinoma cells was examined following the SLC6A8 gene knockdown by transfecting lentivirus vectors harboring SLC6A8 siRNA, which is an efficient and highly specific approach for engineering eukaryotic genomes. 36 - 38 In the knockdown cells, there was a significantly low expression of SLC6A8, thus confirming the efficiency of the partial removal of the gene. Due to the knockdown of SLC6A8 expression by siRNA, proliferation was suppressed, but apoptosis was significantly induced in Huh7 and Hep3B cells.…”