The objective of this study was to investigate the effects of different promoters, DNA vectors, and cationic carriers on transfection in hMSCs from different donors and tissues. The data presented in this work can guide the development of other hMSC transfection systems with the goal of producing clinically relevant, genetically modified hMSCs. Following systematic comparison of each variable, we identified adipose-derived hMSCs transfected with mini-intronic plasmids containing the CMV promoter delivered using Lipofectamine 3000 as the parameters that produced the highest transfection levels. Specifically, we investigated two promoters (cytomegalovirus and elongation factor 1 alpha), four DNA vectors (plasmid, plasmid with no F1 origin, minicircle, and mini-intronic plasmid), two cationic carriers (Lipofectamine 3000 and Turbofect), and four donors of hMSCs from two tissues (adipose and bone marrow) for efficient hMSC transfection. This work takes a complementary approach to addressing the challenges of transfecting hMSCs by systematically investigating key transfection parameters for their effect on transgene expression. However, even with priming, hMSC transfection remains inefficient for clinical applications. To address the shortcomings of nonviral gene delivery to hMSCs, our lab has previously demonstrated that pharmacological “priming” of hMSCs with clinically approved drugs can increase transfection in hMSCs by modulating transfection-induced cytotoxicity. However, gene delivery, particularly through nonviral routes, is inefficient. Human mesenchymal stem cells (hMSCs) are primary cells with high clinical relevance that could be enhanced through genetic modification.
Gene Editing: Technology & Applications.βGlo pAn: The human beta-globin 3’UTR and polyadenylation sequence allows efficient arrest of the transgene transcription.encodes a puromycin N-acetyl-transferase that confers resistance to Puromycin. pac: The pac gene from Streptomyces sp. hph: Resistance to Hygromycin B is conferred by the hph gene from E. Sh ble: Resistance to Zeocin™ is conferred by the Sh ble gene from Streptoalloteichus hindustanus which inactivates Zeocin™ upon binding to the antibiotic. bsr: The bsr gene from Bacillus cereus encodes a deaminase that confers resistance to the antibiotic Blasticidin S. The resistance gene is driven by the SV40 promoter in tandem with the bacterial EM7 promoter to allow antibiotic selection in both mammalian cells and E. CMV enh/prom: The human cytomegalovirus immediate-early gene 1 promoter/enhancer was originally isolated from the Towne strain and was found to be stronger than any other viral promoters.SV40 prom: The Simian Virus 40 promoter allows the expression of the blasticidin resistance gene in mammalian cells.coli origin of replication with the same activity as the longer Ori. SV40 pan: The Simian Virus 40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA.PUNO1: 5’ - Sal I, SgrA I, BamH I, Eco47 III, Nco I, Nhe I - 3’Įach restriction site is compatible with many other enzymes, increasing the cloning options. PUNO: 5’ - SgrAI, SalI, BamHI, Eco47III, PstI, NheI - 3’ The multiple cloning site contains the following restriction sites: The R-U5’ has been coupled to the EF-1α core promoter to enhance stability of DNA and RNA. The EF-1α promoter exhibits a strong activity, higher than viral promoters and, on the contrary to the CMV promoter, yields persistent expression of the transgene in vivo. hEF1/HTLV prom is a composite promoter comprising the Elongation Factor-1α (EF-1α) core promoter and the R segment and part of the U5 sequence (R-U5’) of the Human T-Cell Leukemia Virus (HTLV) Type 1 Long Terminal Repeat.