Integrated DNA Technologies

Transductin™

Documentation and Support

Transductin is a peptide-based transduction delivery reagent for research applications. This technology was developed by Dr. Steven Dowdy at the UCSD School of Medicine and licensed through Traversa Therapeutics, Inc.

Advantages

Avoids the toxicity and immune response often observed with other reagents as Transductin is a peptide-based system that uses the cell's own mechanism for uptake.
Transductin works through a mechanism distinct from cationic lipids and can be used to deliver dsRNAs to most cell lines, including primary cells and ES cells.
Transductin produces fewer off-target effects than cationic lipids and can minimize the risk of triggering an immune response.
Delivery of dsRNA with Transductin is highly reproducible and shows little to no variation from lot-to-lot.
Validated control reagents are available.

OrderingOverviewPerformanceCell Line Dosage Optimization Data

Product Configuration

	Description	Price
Small Lot	2 mg of Transductin	$395.00 USD
Large Lot	10 mg of Transductin	$1,750.00 USD
Trial Kit	1 mg of Transductin HPRT (Human/Mouse/Rat) positive control DsiRNA duplex (1nmole) NC1 negative control DsiRNA duplex (1nmole)	$295.00 USD

Custom Screening lot

Large custom amounts quoted as needed

Place an order now

For custom orders, email custcare@idtdna.com or call 1-800-328-2661 or 319-626-8400 to speak to a Customer Care Representative.

Licensing

Sold under license from Traversa Therapeutics for Buyer's research purposes only.

Transductin is a peptide-based transduction delivery method based on work done by Dr. Steven Dowdy at the UCSD School of Medicine and licensed through Traversa Therapeutics, Inc. Transductin is a small peptide comprised of two Peptide Transduction Domains connected to a Double-Stranded RNA Binding Domain (PTD-DRBD).

Figure 1. Hypothetical structure of Transductin bound to an siRNA duplex (image courtesy of Dr. Steve Dowdy, UCSD).

As shown in Figure 1 above, four PTD-DRBD molecules bind to a single RNA duplex, surrounding the RNA in a circular fashion with a footprint of around 16 bp. For dsRNAs in the size range of 21-27 bp, from 4 to 8 PTD-DRBD groups can associate with a single duplex.

When complexed with siRNAs (unmodified or 2′-OMe modified), Transductin binds to cell surface glycosaminoglycans and is taken up through the process of macropinocytosis where cell surface ruffles become cup-shaped extensions and are then internalized as vesicles. With Transductin, the macropinosome, which contains the siRNA bound to the PTD-DRBD, enters the cell, undergoes a decrease in pH, and releases its contents. This release frees the siRNA to enter the RISC pathway (Figure 2). For more information on the science behind Transductin, see the recent publication from Dr. Dowdy’s lab (Eguchi et al., 2009 Nature Biotechnology 27:567).

Figure 2. Schematic showing the steps involved in the uptake of Transductin via macropinocytosis and delivery of the siRNA cargo into RISC.

Although commonly used cationic lipid reagents can be successful at introducing single and double-stranded oligonucleotides (RNA or DNA) into primary or transformed cell lines, they also suffer from unwanted side effects in certain cell lines, such as toxicity at high concentrations or an immune response within the cell. Transductin utilizes the cell’s own machinery for uptake and so can avoid these unwanted side effects while still achieving a high level of uptake.

Efficient transduction of cells that are hard to transfect or do not respond to other transfection reagents
While many cell lines can be efficiently transfected with a cationic lipid, cell lines such as RAW, MCF7, JAWSII, and PC12 are refractory to most commercially available reagents. When IDT tested MCF7 cells, a maximum of 65% gene knock-down was achieved using lipid-based reagents (Figure 3A), whereas we observed greater than 85% knockdown with as little as 25 nM DsiRNA with 4.5 µM Transductin (Figure 3B).

	Figure 3A	Figure 3B
	Figure 3. MCF7 cells refractory to commercial lipid-based transfection reagents are efficiently transduced with Transductin.
Human breast epithelial carcinoma cell line MCF7 were A) forward transfected with the cationic lipids TriFECTin™, RNAiMAX™, TransIT TKO™ or B) forward transduced with Transductin (4x10⁴ cells) at optimal reagent conditions. 24 hours post transfection, the cells were harvested, RNA was isolated, and cDNA was made. HPRT levels were measured using RT-qPCR with the NC1 negative control levels set at 100%.
IDT also obtained excellent transduction results with RAW, JAWSII, PC12, and a variety of other cell types as shown below in Figure 4.

	Figure 4. Results of knockdown of HPRT in 16 different cell lines using Transductin.
Transduction conditions were optimized for each cell line and an anti-HPRT DsiRNA was introduced using Transductin. HPRT mRNA levels were measured at 24 hours using RT-qPCR as outlined previously.
Reduce the risk of triggering an immune response in susceptible cell types
Some cell lines are deficient in the receptors needed to recognize foreign RNA and are safe to use with any reagents. Other cell lines can respond to synthetic dsRNAs and mount a Type I Interferon response. Use of dsRNAs in primary cells and in vivo presents the greatest risk for this undesired off-target effect. Cationic lipids maximize the likelihood of triggering an immune response by delivering the cargo siRNA into endosomal compartments where critical immune receptors reside. Transductin delivers siRNA via a different mechanism and avoids this compartment and so minimizes the risk of triggering an immune response.
The human brain fibroblast cell line T98G has been shown to mount an immune response when transfected with cationic lipid-based reagents and unmodified DsiRNAs. This induction can be abrogated through the use of 2′-OMe modifications. While evading an immune response is highly desirable, such modifications can lower the potency of the DsiRNA. We find that Transductin can be used to introduce unmodified DsiRNAs into T98G cells without evoking an immune response (Figure 5).

	Figure 5. Transductin does not stimulate interferon inducible gene expression
Human T98G cells were either transfected with the cationic lipid siLentFectTM or transduced with Transductin in a 48-well format. After 24 hours the cells were harvested, RNA was isolated, and cDNA was made. For each gene tested (IFITM1, P56, OAS2, RIG-I), the mRNA levels were normalized to RPLP0 levels with levels in untreated cells set to 1.
High reproducibility with low variation observed from lot-to-lot or well-to-well
Each lot of Transductin is subjected to stringent quality control measures: the purified peptide molecular weight is verified using mass spectrometry, the lot must be free of toxic bacterial lipopolysacharides as determined by the in-vitro HEK-Blue™ LPS Detection system (InvivoGen, San Diego, CA), and the lot must exhibit an 85% or greater knockdown at a 4.5 µM Transductin concentration and 100 nM HPRT-1 DsiRNA in the Neuro2A cell line. The end result is a transduction reagent that gives very consistent results from batch-to-batch. (Figure 6) and well-to-well (Figure 7).

	Figure 6. Consistent reduction of HPRT-1 mRNA levels using different Transductin lots in Neuro2A Cells.
Neuro2A cells were forward transduced with 4.5 µM of Transductin complexed to 300 to 25 nM HPRT-1 DsiRNA, or 300 nM NC1 in a volume of 120 µL in a 48-well polystyrene plate. Cells were plated at a density of 8.5x10⁴ cells/ well 12 hours prior to transduction. 24 hours post-transduction, RNA was isolated and HPRT levels were measured using RT-qPCR with the NC1 negative control levels set at 100%.

	Figure 7. Well-to-well variation in the reduction of HPRT-1 mRNA levels using Transductin in Neuro 2A Cells
Cells were forward transduced with 4.5 µM of Transductin complexed to 100 nM HPRT-1 DsiRNA, or 100 nM NC1 in a volume of 120 µL in a 48-well polystyrene plate. Cells were plated at a density of 8.5x10⁴ cells/ well 12 hours prior to transduction. 24 hours post-transduction, RNA was isolated and HPRT levels were measured using RT-qPCR with the NC1 negative control levels set at 100%.
Transductin is usually used at 3.0 – 6.0 µM concentration with most cell lines. Using our standard 4.5 µM concentration, 1 mg of peptide will transduce 114 wells in a 48 well format plate (using 120 µl medium). More samples can be transduced using 96 well format plates, fewer using 24 well format plates.

Cell Lines Tested With Transductin Using Dicer Substrate HPRT-1 siRNA

For all cell lines tested: cells were forward transduced with 3 to 6 µM of Transductin complexed to 300 to 25 nM HPRT-1 DsiRNA, or 300 nM NC1 in a volume of 120 µL in a 48 well polystyrene plate. Cells were plated at the recommended density 12 hours prior to transduction. 24 hours post-transduction, RNA was isolated and cDNA made with oligo dT and random hexamers using SuperScript II (Invitrogen, Carlsbad, CA). HPRT levels were normalized to RPLPO, RPL23, Odc, or RPL4 levels (Levels were previously determined to be unchanged upon treatment with Transductin) with the NC1 negative control levels set at 100%.

CHO (Chinese Hamster Ovary Carcinoma)