In Vitro Validation of Phosphorodiamidate Morpholino Oligomers

One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-Nucleofector™ or Neon™ electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000™ for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000™ or Lipofectamine 2000™ for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.

Systematic Screening of Commonly Used Commercial Transfection Reagents towards Efficient Transfection of Single-Stranded Oligonucleotides

Non-viral vector-mediated transfection is a core technique for in vitro screening of oligonucleotides. Despite the growing interests in the development of oliogonucleotide-based drug molecules in recent years, a comprehensive comparison of the transfection efficacy of commonly used commercial transfection reagents has not been reported. In this study, five commonly used transfection reagents, including Lipofectamine 3000, Lipofectamine 2000, Fugene, RNAiMAX and Lipofectin, were comprehensively analyzed in ten cell lines using a fluorescence imaging-based transfection assay. Although the transfection efficacy and toxicity of transfection reagents varied depending on cell types, the toxicity of transfection reagents generally displayed a positive correlation with their transfection efficacy. According to our results, Lipofectamine 3000, Fugene and RNAiMAX showed high transfection efficacy, however, RNAiMAX may be a better option for majority of cells when lower toxicity is desired. The transfection efficacy of Lipofectamine 2000 was compromised by its high toxicity, which may adversely affect its application in most cells. We firmly believe that our findings may contribute to the future In vitro delivery and screening of single-stranded therapeutic oligonucleotides such as antisense oligonucleotides, antimiRs, and DNAzymes.

205 Optimization of Transfection Efficiency for CRISPR/Cas9-Induced Genomic Editing in Porcine Fibroblast Cells

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system creates DNA double-stranded breaks (DSB) at specific sequences and allows efficient genomic modification, even in species previously resistant to gene editing. The DSB can be repaired using non-homologous end joining (creating insertions/deletions) or by homology directed repair (HDR) using a donor DNA with small changes at the cut site, giving rise to precise targeted modifications. Despite growing interest in genome editing using RNA-guided endonucleases, the efficiency of HDR is only 0.5 to 20%. The objective of this study was to optimize transfection conditions in order to increase efficiency of HDR for CRISPR/Cas9 targeted genomic editing of porcine cells. We utilised the Swedish mutation of the porcine APP gene causing early-onset Alzheimer’s disease. We first tested co-transfection of 2 plasmids, one containing our guide RNA (gRNA) and another containing the Cas9 nuclease, using square-wave electroporation. Upon analysis via T7 endonuclease assay I, this method failed to produce a DNA DSB at the target site. Next, we tested transfection of a single vector containing both the gRNA and Cas9 nuclease. Three gRNAs targeting exon 17 of the porcine APP gene were constructed and inserted into CRISPR/Cas9 pGuide-it plasmids expressing green fluorescent protein (GFP). Plasmid DNA was transfected into cultured porcine fibroblast cells by 3 methods: Lipofectamine 2000, square-wave electroporation, and exponential-wave electroporation. To determine which method yielded the highest transfection rates, cells were analysed using flow cytometry to detect GFP expression. The transfection efficiency, percentage of cells expressing GFP, was analysed by one-way ANOVA and individual pair wise comparisons. Twelve microliters of Lipofectamine 2000 per well of a 6-well plate with 200 ng of plasmid DNA per μL of Lipofectamine was used to optimize transfection rates, as suggested by the manufacturer. Removal of transfection media after 48 h yielded higher transfection rates than removal after 24 h (6.9% ± 0.7 v. 2.2% ± 0.1; P = 0.02). For electroporation, 12.5 and 25 μg of plasmid DNA per mL was added to 0.2- and 0.4-mm gap cuvettes, respectively, each containing cell suspensions of 1 × 106 cells mL−1. Square-wave electroporation was performed at 300 V for three 1-ms pulses in 0.2-mm cuvettes. Exponential-wave electroporation was performed at 350 V and 500 μFD in both 0.2-mm and 0.4-mm cuvettes. Exponential-wave electroporation containing 25 μg of plasmid DNA/mL of cell suspension yielded the highest average transfection efficiency, 22.8% (P < 0.00001), compared with square-wave electroporation and transfection using optimized Lipofectamine 2000 conditions (9.1 and 1%, respectively). All 3 gRNAs resulted in similar transfection rates. In conclusion, efficiency of transfection of the CRISPR/Cas9 system into porcine cells is optimized using exponential-wave electroporation of a single plasmid CRISPR system.