Broad spectrum DNA transfection system: TransIT-X2

TransIT-X2® from Mirus Bio is a low toxicity, dynamic transfection system for the superior transfection of plasmid DNA, siRNA, miRNA and CRISPR/Cas9 components into mammalian and primary cells. Compatible with adherent and suspension cells, the animal-origin free TransIT-X2® achieves both exceptional transient and stable transfections.

Benefits of using TransIT-X2
• Novel nonliposomal, polymeric delivery
• Efficient delivery of plasmid DNA, siRNA/miRNA and CRISPR/Cas9 components
• Peak performance in a broad range of cell types
• Low cellular toxicity
• Animal-origin free
• Serum compatible
• DNA and siRNA cotransfection compatible
• Adherent and suspension cell compatible
• Suitable for multiple applications

TransIT-X2 v Lipofectamine® 2000

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 ‡.

Visualisation of high GFP expression using TransIT-X2

4-8 µl TransIT-X2™ used to transfect 2 µg plasmid DNA encoding EGFP into A549, CHO-K1, HepG2, LNCaP, MDCK, PC12, primary human mammary epithelial cells (HMEC) and normal human dermal fibroblasts (NHDF). Images (32X) captured at 48 hours post-transfection.

Functional cotransfection of plasmid DNA and siRNA using the TransIT-X2

TransIT-X2 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.

TransIT-X2 achieves higher knockdown than Lipofectamine® 2000

TransIT-X2 and Lipofectamine® 2000 were used to transfect siRNA targeting endogenous proteins – GAPDH and AHA1 or to deliver a nontargeting siRNA control in NHDF. Cells were transfected 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.

TransIT-X2 outperforms Lipofectamine® for RNP delivery

Ribonucleoprotein (RNP) complexes composed of PPIB (cyclophilin B) targeting two part gRNA (IDT) and Cas9 protein (PNA Bio) were delivered into HEK293T/17 and U2OS cells using TransIT-X2® Dynamic Delivery System or Lipofectamine® CRISPRMAX™ or Lipofectamine® RNAiMAX or Lipofectamine® 3000 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. A T7E1 mismatch detection assay was used to measure cleavage efficiency at 48 hours post-transfection.

Applications
• Stem cell transfection
• Stable transfection
• CRISPR/Cas9 genome editing
• Cotransfection
• Gene knockdown

TransIT-X2 citations

Free samples
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Material available for download
TransIT-X2 protocol
TransIT-X2 MSDS
TransIT-X2 protocol optimisation note
Optimisation of DNA, RNA and RNP delivery methods for efficient CRISPR/Cas9

Learn more about using TransIT-X2 for CRISPR/Cas9 genome editing

Lipofectamine, Alexa Fluor and CRISPRMAX are registered trademarks of Life Technologies , Inc.