TransIT-X2 Dynamic Delivery System – 0.3 mL

Supporting Data

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 ‡.

Resource: Cell-type specific transfection protocol recommendations (PDF).

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.

Visualization of High GFP Expression Using TransIT-X2® Dynamic Delivery System. TransIT-X2® Dynamic Delivery System was used to transfect plasmid DNA encoding EGFP into A549, CHO-K1, HepG2, LNCaP, MDCK, PC12, primary human mammary epithelial cells (HMEC) and normal human dermal fibroblasts (NHDF). Transfections were performed in 35 mm MatTek dishes using 4-8 µl of TransIT-X2® to deliver 2 µg of DNA. Images (32X) were captured at 48 hours post-transfection using a Zeiss Axiovert S100 inverted fluorescence microscope.

* indicates primary cell types

High GFP Transfection Efficiency in Multiple Cell Lines and Primary Cells Using TransIT-X2® Dynamic Delivery System. TransIT-X2® Dynamic Delivery System was used to transfect plasmid DNA encoding EGFP into A549, CHO-K1, Hep G2, MDCK, LNCaP, PC-12, primary human mammary epithelial cells (HMEC) and normal human dermal fibroblasts (NHDF). Transfections were performed in 96-well plates using 0.2-0.4 µl of TransIT-X2® to deliver 0.1 µg of DNA (2:1, 3:1 or 4:1 reagent: DNA ratio). Triplicate wells were assayed 48 hours post-transfection using guava easyCyte™ 5HT Flow Cytometer.

*indicates primary cell types

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).

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.

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.

Efficient Genome Editing with Cas9 Plasmid DNA + Guide RNA Oligonucleotides. HEK293T/17, U2OS and NHDF cells were co-transfected with 0.5 µg of Cas9 encoding pDNA (MilliporeSigma) and 50nM PPIB targeting 2-part gRNA (Dharmacon) using TransIT-X2® Dynamic Delivery System (2 µl/well of a 24-well plate, Mirus Bio).  A T7E1 mismatch detection assay was used to measure cleavage efficiency at 48 hours post-transfection.

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.

ation present at position 705 of the ß-globin intron activates two cryptic splice sites within the intron that lead to the production of a spliced luciferase mRNA that is disrupted by a small intron with an in-frame stop codon, thus preventing translation of functional luciferase protein. The transfection of a 2’OMe oligonucleotide (TriLink BioTechnologies, Inc.) complementary to the cryptic 705 splice site inhibits splicing at the cryptic splice sites enabling the complete removal of the ß-globin intron and production of a mRNA with a complete, uninterrupted luciferase ORF.

The HeLa-Luc 705 cell line was transfected with increasing amounts of the anti-705 splice site 2’OMe RNA oligo at the indicated final concentrations using the TransIT®-Oligo Transfection Reagent. The cells were harvested 24 hours post-transfection and assayed for luciferase activity. The increase in luciferase activity indicates effective delivery of the anti-705 splice site RNA oligo using the TransIT®-Oligo Reagent.