Baculovirus-induced recombinant protein expression in human mesenchymal stromal stem cells: A promoter study

An improved Tet-on system in microRNA overexpression and CRISPR/Cas9-mediated gene editing

“…However, the fold induction of P PGK was the optimal among them. Promoter activities are believed to be conserved within mammalian tissues [31, 32], which was confirmed in this study that P PGK is consistently dominant in multiple human and mouse cell lines. CMV promoter was an exception in that it showed variability from one cell type to another.…”

Abstracts from the 25th European Society for Animal Cell Technology Meeting: Cell Technologies for Innovative Therapies

“…For achieving a baculovirus-induced differentiation of hMSCs, the promotor and the expression strength of the recombinant protein are crucial factors. Still, there are still few comparative promotor studies [17,18]. However, a successful virus uptake is the prerequisite for a successful protein expression.…”

“…The experimental design comprises a two level factorial screening, set-up using Design Expert V9.For the transduction 60,000 c/cm 2 were seeded in 24-well plates with DMEM + 10% FCS and incubated overnight at 37°C, 8% CO 2 and humidified atmosphere. The recombinant baculovirus using an integrated EF1α promoter to control GFP expression, described elsewhere [18], was diluted to the respective concentrations in the different surrounding fluids. After discarding the cultivation medium of the hMSC-TERT, 1 mL of virus containing solution was added to the cells.…”

Baculoviruses in Gene Therapy and Personalized Medicine

“…63,64 Comparatively, in human mesenchymal cells, often the focus of regenerative medicine, human cytomegalovirus, ubiquitin C, phosphoglycerate kinase, and elongation factor-1 alpha (EF1α) promoters have been incorporated into the Bac-to-Bac system. 65 Particularly, EF1α demonstrated the highest transgene expression indicating the efficiency of the promoter is largely dependent upon cell type and more importantly revealing the potential for stem cell gene therapy. Moreover, promoters can be used in combination with transcriptional enhancers to increase transgene expression.…”

“…Berger et al incorporated an array of small synthetic DNA plasmids termed acceptors and donors. 25 The acceptors can be loaded with several genes to produce eukaryotic protein complexes with many subunits, termed Addition of gp64 and thogotovirus glycoproteins 4 to 12-fold increase in transgene expression [53] Viral surface display Addition of VSVG 10 to 100-fold increase in transgene expression [58] Addition of influenza virus neuraminidase Expression on both the infected cells and budded virus [54] Addition of antibody fragments (Fc IgG) Bound specifically Fc gamma receptors on antigen-presenting cells [55] Addition of SeMNPV F protein Enhanced tropism for vertebrate cells [56] Addition of tumor-homing peptides with VSVG 2 to 5-fold increase in transgene expression [59] Display avidin to take advantage of the avidin-biotin interaction 5-fold increase in transduction efficiency in rat malignant glioma cells and a 26-fold increase in rabbit aortic smooth muscle cell [60] Cytoplasmic transduction peptide fused to gp64 and Bac VP39 fused to HIV Tat protein 0.4 to 5-fold increase in transgene expression [61] Circumsporozoite protein variants and thrombospondin-related anonymous protein Specific and enhanced transduction efficiency of hepatocytes [62] Promoter selection Viral polyhedrin and p10 promoter Improved quality and quantity of protein expression [17] Viral promoter p6.9 with hr3 and repeated burst sequences Altered gene expression to occur one day earlier with 94 times greater transgene expression [66] Viral promoter 39 Improved transgene expression and protein folding [14] Immediate early gene (IE1) promoter Produced continuous and stable protein with more efficient complex human glycoprotein [63] Basic juvenile hormone-suppressible protein 2 (pB2) Increased recombinant protein expression earlier than polyhedrin alone [64] Human cytomegalovirus (CMV) promoter Lower transgene expression when compared to CAGG and EF1α [65] Human ubiquitin C promoter (UBC) Lower transgene expression when compared to CAGG and EF1α [65] Chicken β-actin promoter coupled with CMV early enhancer (CAGG)…”