Table of Contents
- Why Choose Morpholinos?
- What is a Morpholino?
- Why should I use Morpholino oligos for my antisense experiments?
- How do the properties of Morpholinos compare to other antisense structural types?
- Can Morpholino oligos be used as probes?
- Choosing the Optimal Target (design of oligo)
- What length oligo should give me the highest antisense activity?
- Can I order an oligo longer than 25-mer?
- What end modifications do you offer?
- Can I add my own label or molecule to a Morpholino oligo?
- Can I target point mutations with Morpholino oligos?
- Can I target the amino acid coding sequence of an mRNA with Morpholinos?
- Can Morpholinos block mRNA processing?
- Using Morpholinos: Methods and Protocols
- What do you do for purification?
- Will Gene Tools further purify my oligo?
- Can Morpholino oligos be used as PCR primers?
- Can Morpholino oligos enter unperturbed tissue culture cells?
- What is the best method for delivering Morpholino oligos into adherent cells?
- How do I deliver Morpholino oligos into non-adherent cells?
- Can I use lipofectin or other cationic lipids to deliver Morpholino oligos?
- What cell types can I use with Morpholino oligos?
- What is the level of inhibition one can expect from Morpholino oligos?
- Ordering Products
- How much do Morpholino oligos cost?
- Do I get a discount if I order more oligos?
- Are there extra costs associated with international orders?
- Can I order less than 300 nmol?
- Can I order more than 300 nmol?
- Do you offer a standard control oligo at a reduced price?
- After ordering, how long until I receive my Morpholino oligos?
Why Choose Morpholinos?
- What is a Morpholino? Morpholino oligos get their name from the morpholine rings in their backbones, which replace the ribose or deoxyribose rings characteristic of RNA and DNA type oligos. Morpholinos contain uncharged phosphorodiamidate intersubunit linkages instead of the anionic phosphodiester linkage found in natural nucleic acids. The morpholine rings carry A, C, G or T bases positioned suitably for Watson-Crick base pairing.
- Why should I use Morpholino oligos for my antisense experiments? Because you want valid results the first time (see also why choose Morpholinos ). There is no other gene knockdown reagent (including siRNA, PNA, mPNA, S-DNA, and LNA) that combines the properties of stability, nuclease-resistance, efficacy, long-term activity, water-solubility and exquisite specificity. Only Morpholino oligos provide all of these.
In contrast to RNase-dependent or RISC-dependent reagents, Morpholino oligos may be targeted either to block translation or to alter nuclear processing. Morpholinos targeted against splicing provide these advantages over translation blocking oligos:
- End products that are quantifiable with RT-PCR
- Abolishes requirement for antibodies
- Enables targeting of specific messengers
- Can block expression of splice-variants
- Can elucidate function of protein domains
Most Morpholino oligos selected according to our targeting guidelines achieve good to excellent efficacy and excellent specificity.
- carboxyfluorescein or lissamine (fluorescent tags);
- dabcyl (fluorescence quencher);
- biotin (affinity tag);
- or primary amine (functional group for reaction);
Choosing the Optimal Target (design of oligo)
- What length oligo should give me the highest antisense activity? The longer the oligo the greater the efficacy. Therefore we recommend ordering 25-mers, our longest available oligo.
- Can I order an oligo longer than 25-mer? Yes. Our current 25-mer Morpholinos generally provide higher efficacies than any other commercially-available structural type, but for a significant increase in price we can make them even longer.
- What end modifications do you offer? We offer the following end modifications: lissamine (sulforhodamine B), a fluorescein (carboxyfluorescein), dabcyl, a primary amine, and biotin (see structures).
- Can I add my own label or molecule to a Morpholino oligo? Yes. Our standard Morpholino has a moderately nucleophilic secondary amine (a Morpholine nitrogen, pKa about 6.4) on its 3' terminus. This amine can be acylated under suitable conditions. Our oligos can be modified with a primary amine on either the 3' or 5' end. Either of these primary amines are more reactive than the secondary amine.
- Can I target point mutations with Morpholino oligos? No. The property which gives Morpholino oligos their spectacular sequence specificity (a large MIL value) also largely precludes their use for effectively targeting point mutations. However, Morpholinos are the best tools for studying point mutations, polymorphisms, and a host of other genetic variations using the gene switch strategy or splice-blocking Morpholinos.
- Can I target the amino acid coding sequence of an mRNA with Morpholinos? Because Morpholino oligos functions solely by a steric blocking mechanism, only the leader sequence and the first 25 bases of the amino acid coding region can be targeted for translation blocking applications (see: targeting guidelines).
- Can Morpholinos block mRNA processing? Yes. Morpholinos targeted at the splice donor site or exon/intron boundary of internal exons predictably block splicing events.
Using Morpholinos: Methods and Protocols
- What do you do for purification? Waste products (synthesis resin, ammonia, cleaved base-protective groups, and minor amounts of short truncation fragments) are removed by selective precipitation. After spectrophotometric quantitation and MALDI-TOF mass spectral analysis, the product is freeze dried and sterilized to give 300 nmol of salt-free non-ionic oligo.
- Will Gene Tools further purify my oligo? No. Further purification has been found to afford no significant improvement in efficacy, but further chromatographic purification can seriously compromise specificity due to cross-contamination with small amounts of other oligos previously purified on the same chromatography column.
- Can Morpholino oligs be used as PCR primers? No. There is no advantage over using DNA as a primer, Morpholinos have no 3'-OH for primer extension, and the Morpholino sequences can't be used as a template for DNA synthesis.
- Can Morpholino oligos enter unperturbed tissue culture cells? Apparently yes, but high concentrations (tens of µM) and long incubation times (multiple days) are required to achieve even very modest amounts of delivery in most cell types (see Suwanmanee et al. for details). With their covalently-attached delivery moiety, Vivo-Morpholinos can efficiently enter most cultured cell types without requiring a secondary delivery technique.
- What is the best method for delivering Morpholinos into adherent cells? Scrape Delivery is a simple, fast, and effective procedure for delivering Morpholino oligos into adherent cells (see: Patridge et al., 1996)
- delivers your product into essentially 100% of the cells in many cell types
- can be used with cells in small culture plate wells (i.e. 24, 48 or 96 well plates); and
- delivers product into poorly-adherent cells or cells in suspension.
- How do I deliver Morpholino oligos into non-adherent cells? Vivo-Morpholinos are the simplest and most effective method for delivering oligos into cells grown in suspension.
- Can I use lipofectin or other cationic lipids to deliver Morpholino oligos? Investigators have reported delivering Morpholinos with cationic lipids, but non-ionic Morpholinos will not complex with cationic lipids. Gene Tools offers more effective delivery methods.
- What cell types can I use with Morpholino oligos? Morpholinos should be effective in all cell types into which they can be delivered.
- What is the level of inhibition one can expect from Morpholino oligos? In cell free translation systems, complete inhibition of translation is typically achieved at 1 µM or less.
The Endo-Porter delivery reagent is our reagent of choice for delivering ummodified or tagged Morpholinos to cultured cells as you can vary oligo dose and deliver multiple oligos simultaneously when using Endo-Porter.
Vivo-Morpholinos are the most convenient option for most cell types, as they are simply added to the culture medium to effect delivery into cultured cells.
If electroporation is tolerated by a particular cell type, then it is an efficient method for delivery of Morpholinos into cultures. Concentration of cells by centrifugation prior to electroporation allows smaller volumes of Morpholino solution to be used, conserving oligo.
Endo-Porter can be used in suspension cells with unmodified or tagged Morpholinos. Because the Endo-Porter tends to settle to the bottom of wells, the cultures should be gently swirled during incubation to keep the Endo-Porter particles suspended so they can contact the suspension cells.
With Scrape Delivery in cultured cells, complete inhibition of translation is typically achieved at an extracellular concentration of 10 µM or less.
With Endo-Porter in cultured cells, complete inhibition of translation is typically achieved at an extracellular concentration of 5 µM.
- How much do Morpholino oligos cost? The prices of all our products, including custom Morpholino oligos, can be found on our Current Price List.
- Do I get a discount if I order more oligos? Prices of Morpholino oligos were reduced in 2004 and price breaks are no longer available on the 300 nmol package size of custom Morpholino oligos. However, larger quantities of a single sequence are available at a reduced per nmol cost.
- Are there extra costs associated with international orders? Yes. International customers will be responsible for duties, taxes and tariffs imposed on the oligo shipment by the customer's country (click for details).
- Can I order less than 300 nmol? No. Our synthesizers are currently set up to produce 300 nmol, 1000 nmol and 6000 nmol batch sizes. Small quantities (50 nmol) of some individual zebrafish targeted Morpholinos are available from Open Biosystems, Inc.
- Can I order more than 300 nmol? Yes. 1,000 nmol and 6,000 nmol sizes (delivered quantity) are available.
- Do you offer a standard control oligo at a reduced price? Yes. We offer a Standard Control Morpholino oligo 25-mer; please refer to our price list for current pricing.
- After ordering, how long until I receive my Morpholino oligos? It usually takes 7 to 10 business days to prepare, process and ship a custom Morpholino order. Vivo-Morpholinos take a day or two more. If an oligo fails quality control, it will add time (usually about a week) before the order is shipped; QC failure is uncommon (a few percent of oligos). Orders shipped within the United States are sent via 2-day FedEx service (overnight shipping is available). For international researchers extra transit time may be required to clear customs in your country.