TransIT-X2^®

Fig. 1 | TransIT-X2^® System Enables Superior Gene Expression in a Variety of Cell Types. TransIT-X2^® Transfection Reagent Enables Superior Transfection in a Variety of Cell Types. TransIT-X2^® Dynamic Delivery System and Lipofectamine^® 2000 Transfection Reagent were used to transfect plasmid DNA encoding luciferase into 41 different cell types at three reagent-to-DNA ratios. Luciferase expression was compared at 24 hours post-transfection using a standard luciferase assay. Head-to-head comparisons at optimized ratios illustrate superior or equal luciferase expression using TransIT-X2^® in 36 of 41 cell types; 17 cell types that had expression levels 2-fold higher are denoted with ‡.

Fig. 2 | TransIT-X2^® Dynamic Delivery System Outperforms Lipofectamine^® Reagents. A549 (A) or MDCK (B) cells were transfected with luciferase encoding plasmid DNA using either TransIT-X2^® (Mirus Bio), Lipofectamine^® 2000 (Thermo Fisher Scientific) or Lipofectamine^® 3000 (Thermo Fisher Scientific) for 24 hours at indicated reagent-to-DNA ratios or reagent-to-P3000™-to-DNA ratio. Transfection was measured by luciferase activity using a conventional assay. Cytotoxicity was assessed by quantifying the LDH released from the cytosol of damaged cells compared to cells alone. Higher transgene expression and lower cytotoxicity was observed in cells transfected with TransIT-X2^® at optimal ratios compared to cells transfected with Lipofectamine^® 2000 or Lipofectamine^® 3000.

Fig. 3 | Functional Co-transfection of Plasmid DNA and siRNA Using the TransIT-X2^® Dynamic Delivery System. TransIT-X2^® Dynamic Delivery System was used to transfect plasmid Cy^®5 labeled DNA encoding nuclear YFP and Cy^®3 labeled siRNA into HeLa cells. Transfection was performed in 6-well plates with Poly-L-Lysine (PLL) coated coverslips using 4 µl of TransIT-X2^® to deliver 2 µg of DNA and 25 nM siRNA (2:1 reagent:DNA ratio). Actin cytoskeleton was stained using Alexa Fluor^® 350 Phalloidin. Images (63X) were captured at 24 hours post-transfection using a Nikon A1R confocal microscope. Image key: yellow (nuclear YFP), blue (Cy^®5 labeled DNA), red (Cy^®3 labeled siRNA), green (actin cytoskeleton).

Fig. 4 | TransIT-X2^® Dynamic Delivery System Achieves Higher Knockdown than Lipofectamine^® 2000. TransIT-X2^® Dynamic Delivery System and Lipofectamine^® 2000 Transfection Reagent were used to transfect siRNA targeting endogenous proteins – GAPDH and AHA1 or to deliver a non-targeting siRNA control in normal human dermal fibroblasts (NHDF). Cells were transfected in a 6-well plate using 4 µl of TransIT-X2^® or 6 µl of Lipofectamine^® 2000 and 25 nM siRNA according to each manufacturer's protocol. The amount of GAPDH or AHA1 mRNA was measured relative to 18s rRNA levels using qRT-PCR and then normalized to the mRNA levels of the non-targeting control, 48 hours post-transfection. Error bars represent the standard deviation of triplicate wells.

Fig. 5 | Effective miRNA Delivery Using TransIT-X2^® Dynamic Delivery System Yields Decreased Levels of PTK9 mRNA. TransIT-X2^® Dynamic Delivery System and Lipofectamine^® 2000 Transfection Reagent were used to transfect Pre-miR™ hsa-miR-1 miRNA Precursor or mirVana™ miRNA mimic, miR-1, both known to decrease PTK9 mRNA levels. A Pre-miR™ negative control was also transfected to assess baseline mRNA levels. T47D cells were transfected in a 12-well plate using 3 µl of TransIT-X2^® or Lipofectamine^® 2000 and 50 nM miRNA according to each manufacturer's protocol. The amount of PTK9 mRNA was measured relative to 18s rRNA levels using qRT-PCR and then normalized to the mRNA levels of the negative control, 48 hours post-transfection. Error bars represent the standard deviation of triplicate wells.

Fig. 6 | TransIT-X2^® Outperforms Lipofectamine^® for RNP Delivery. Ribonucleoprotein (RNP) complexes composed of PPIB (cyclophilin B) targeting 2-part gRNA (IDT) and Cas9 protein (PNA Bio) were delivered into HEK293T/17 and U2OS cells using TransIT-X2^® Dynamic Delivery System (1 µl/well, Mirus Bio) or Lipofectamine^® CRISPRMAX™ (1.5 µl/well and 1 µl/well of Lipofectamine^® Cas9 Plus™ Reagent, ThermoFisher) or Lipofectamine^® RNAiMAX (1.5 µl/well, ThermoFisher) or Lipofectamine^® 3000 (1.5 µl/well and 1 µl/well of P3000™ Reagent, ThermoFisher) in a 24-well format according to the manufacturers’ protocol. Varying levels of gRNA (6 nM or 12 nM) were tested with 6 nM Cas9 protein (PNA Bio). A T7E1 mismatch detection assay was used to measure cleavage efficiency at 48 hours post-transfection.