Special Delivery Morpholino oligos provide substantially improved delivery into the cytosol/nuclear compartment of both adherent and suspension cells compared to previous scrape and osmotic delivery methods. While Gene Tools no longer recommends the Special Delivery system as the best available reagent-based culture delivery system for Morpholinos, a protocol for making your own heteroduplexes is available here, as well as the protocol for delivering the heteroduplexes into cultured cells. Gene Tools still manufactures Special Delivery oligos by arrangement. The EPEI delivery solution is still available by request from Gene Tools.

The Special Delivery formulation consists of two components. One component is a pre-paired heteroduplex of Morpholino oligo and partially complementary DNA oligo. The other component is a weakly basic delivery reagent, ethoxylated polyethylenimine (EPEI), delivered as a green solution.

Delivery entails three simple steps:

1. Mix the Morpholino/DNA component with EPEI and incubate at room temp. 20 minutes.
2. Add the Morpholino/DNA/EPEI solution to your cells.
3. Three hours later remove the solution and replace with fresh culture medium.

The first step results in electrostatic binding of the anionic Morpholino/DNA duplex to cationic sites of the EPEI. Step 2 results in the electrostatic binding of the weakly cationic Morpholino/DNA/EPEI complex to anionic cell surfaces leading to endocytosis of the complex. Subsequent acidification within the endosome protonates additional weakly basic nitrogens of the EPEI, and this more extensively ionized EPEI permeabilizes the endosomal membrane causing release of the Morpholino oligo into the cytosol/nuclear compartment of the cell.

A related polyethylenimine reagent called ExGen 500 has been shown to be effective in delivering DNA into a variety of widely used tissue culture cell types. The figure below compares delivery by our special delivery formulation to that of scrape delivery, osmotic delivery, and delivery using ExGen 500.

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1. Preparing Stock Solutions
To one vial containing 300 nmol of lyophilized Special Delivery Morpholino/DNA add 600 µL sterile water to make a 0.5 mM stock solution. To one vial containing 100 nmol of Special Delivery Standard Control Morpholino oligo add 200 µL sterile water to make a 0.5 mM stock solution. The EPEI Special Delivery Solution is 200 µM in EPEI.
2. Materials You Supply For Delivery
1. Sterile 15.0 mL capped tubes
2. Sterile water
3. Non-adherent or adherent cells preferably at 80-100% confluency in the flask/dish of choice
4. Prewarmed serum-free and serum-containing media for the cells you study
3. Protocol for treating cells in 3 wells of a 24-well culture plate
1. To a sterile 15 mL centrifuge tube:


1. Add 188.8 µL H₂O
2. Add 5.6 µL of the 0.5 mM Special Delivery Morpholino/DNA stock solution
3. Mix
4. Add 5.6 µL of the 200 µM EPEI Special Delivery solution
5. Vortex immediately and let stand at room temperature for exactly 20 minutes


2. After 20 minutes incubation at room temp, add 1.8 mL of serum-free medium and vortex immediately to generate the complete delivery solution.


3. Remove media from cells (requires centrifugation in the case of non-adherent cells) and then add 500 µL of the complete delivery solution to each of the 3 wells, briefly mix, and return to the incubator.


4. After incubation for 3 hours, remove complete delivery solution from cells and replace with fresh serum-containing medium (centrifugation is required for non-adherent cells).

Cells can be assayed as soon as 16 hours after media replacement. Morpholino oligos are stable and totally nuclease resistant so there is no need to re-deliver Morpholinos within the first few days. In some cases turnover of the targeted protein may be slow so incubation for several days may be required to significantly reduce the level of previously synthesized protein. However, when cells are allowed to undergo more than 5-10 divisions it may be necessary to treat again to compensate for the dilution of oligo due to cell divisions.
4. Table for scaling the above protocol for triplicate samples in selected plates and flasks

Solutions	48 Well Plate	24 Well Plate	12 Well Plate	6 Well Plate	25 cm2 Flask	75cm2 Flask
H2O	94.9 µL	188.8 µL	283.2 µL	566.4 µL	1.0 mL	3.4 mL
Morph/DNA Stock	2.8 µL	5.6 µL	8.4 µL	16.8 µL	33.6 µL	100.8 µL
EPEI Special Delivery solution	2.8 µL	5.6 µL	8.4 µL	16.8 µL	33.6 µL	100.8 µL
Serum-free medium	0.9 mL	1.8 mL	2.7 mL	5.4 mL	10.8 mL	32.4 mL
Aliquot per well or flask (3 samples)	0.25 mL	0.5 mL	0.75 mL	1.5 mL	3.5 mL	11 mL

5. Storage of Reagents
The EPEI Special Delivery Solution should be stored tightly capped at 4C. The Morpholino/DNA stock solution should be stored at 20C. Freezing and thawing of the Morpholino/DNA stock solution should not adversely affect results.
6. Troubleshooting and Optimization
1. The EPEI Special Delivery Solution is killing more than 40% of the cells.

First make sure that you used the right amount of EPEI Special Delivery Solution for your scaled experiment (see table above). Second, make sure you are not using cells that were confluent more than a day. Such cells tend to be very sensitive to changes in conditions. Finally, you can reduce the Morpholino/DNA stock and EPEI stock together by as much as 1/3 and still achieve respectable delivery. For example with 24 well plates (see table above) you could reduce the volumes of the Morpholino/DNA stock and EPEI Special Delivery solution to 3.7 µL each. We have found for particularly sensitive cells (i.e. neuroblastoma and MDAMB231 cells) that reducing the EPEI volume to 4.0 µL and increasing the Morpholino/DNA volume to 10.0 µL has not only eliminated toxicity but resulted in significant delivery. Since the product has not been tested in all cell types, it is possible that your cells are not amenable to delivery by EPEI Special Delivery Solution. In such an event, if the cells are adherent you can utilize scrape delivery to achieve respectable results. The DNA partially paired to Morpholino oligo does not interfere with scrape delivery of the Morpholino oligo.


2. Can I optimize delivery for the cells I study?

You may be able to achieve better delivery by varying the concentration of EPEI in your preparations, but excessive EPEI may result in increased toxicity. We suggest that you use the Morpholino/DNA amounts listed in the table above, but vary the EPEI volumes in 10% increments (change H₂O volumes accordingly). Alternatively you can vary the Morpholino/DNA and EPEI Special Delivery solution by equivalent 10% increments. Watch for toxicity. You may wish to accept some cell toxicity in order to achieve greater delivery.


3. Shouldn't I use EPEI without oligos as a delivery control?

The presence of Morpholino/DNA heteroduplexes complexed with the EPEI decreases the toxicity of the EPEI. Using uncomplexed EPEI as a delivery control will kill lots of cells. A good choice for a delivery control is the Special Delivery standard control Morpholino, which produces all of the effects on the cells of complexed EPEI delivery without the antisense effect of your experimental oligo.


4. Successful delivery has been reported for the following cell types:
• HeLa cells
• NIH3T3
• All fibroblast cell lines
• All undifferentiated colon carcinoma cell lines
• Primary hepatocytes
• HL60 human leukemia cell line
• BHK cells
• PC3 prostate cell line
• MCF7 cells
• 293 cells
• p19 embryonic carcinoma cell line
• THP1 macrophage and rho 274.1 macrophages
• MDAMB231 cells
• Primary endothelial cells