What information do you need from me to produce my vector?
Please download our Requirements for Service form. This form needs to be completed for us to manufacture your custom vector. Please include the following information:
- AAV serotype to be produced.
- Promoter name and source (e.g., human, mouse, viral, etc.).
- Transgene name and source (e.g., human, mouse, etc.).
- Biological activity of transgene.
- PolyA sequence (if applicable).
- Other significant map details, such as intron, enhancer, etc.
How do I prepare my AAV cis plasmid for shipping?
- Maintain the plasmid in a recombinase minus (rec-) cell line (e.g. stbl2, SURE).
- Purify it using an endotoxin-free kit.
- Send at least 1200 μg of plasmid packaged with ice packs, with the tube well shielded from breakage.
- Verify the integrity of the inverted terminal repeats (ITRs) using a SmaI or XmaI digest. The tertiary structure of the ITRs makes sequencing through them extremely difficult, so SmaI digests are used to verify that the ITRs have not been lost or corrupted during the cloning and expansionstages.
- Verify the integrity of the expression cassette using restriction enzyme mapping and/or sequencing.
- Optional, but recommended: Do a transient transfection of your plasmid into an appropriate cell line to confirm transgene expression and/or function.
What are the typical lead times?
REGENX requires six to eight weeks from receipt of the AAV cis plasmid to shipment of your purified rAAV preparation.
If you prefer to send REGENX your cDNA or expression cassette to clone into the AAV cis plasmid, it will take approximately 12 weeks to return your purified rAAV preparation.
What is the typical titer of a vector preparation?
The rAAV prep will usually contain at least 1×1013 GC (genome copies) in 1.5-2.0 ml of volume for reporter constructs and at least 5×1012 GC (genome copies) in 1.5-2.0 ml for custom vectors.
We prefer to send back your prep in its concentrated form (rather than at a pre-set titer) so that you can choose the appropriate dilution level before use.
Can you send me a cis cloning plasmid that I can use to create the vector plasmid?
Yes, we have cis cloning plasmids into which you can clone your cDNA or your expression cassette. We have plasmids with a variety of promoters (see below).
Do you need to know what gene I want to use?
Yes, it is a requirement of the local biosafety committee. However, be assured that the information will be held in the strictest confidence, and will never be shared with any other customers.
What are the limitations on the transgene?
- The total amount of sequence that can be packaged into an AAV is approximately 4.8 kb.
- The ITRs take up approx. 300 bp, leaving about 4.5 kb of foreign sequence to be inserted.
- The exact size of transgene depends on the size of your promoter, polyA, and any other sequences you include (such as an intron).
What promoters are available, and are they tissue-specific?
REGENX offers the following promoters:
- CB: chick beta-actin promoter with CMV enhancer
- CMV: human cytomegalovirus immediate-early enhancer/promoter
- TBG: liver-specific promoter
- Additional promoters specific for muscle, heart and other tissues are available on request.
Please contact us for additional information.
Can you provide vectors with standard reporter genes?
REGENX can provide vectors with eGFP, TurboRFP (red), ZsGreen, YFP (yellow), alkaline phosphatase, alpha-1-antitrypsin (A1AT), cytoplasmic LacZ (β-galactosidase), and nuclear LacZ reporter genes.
Can I use self-complementary AAV (scAAV)? If so, what are the limitations?
scAAV (also known as double-stranded AAV (dsAAV)) is produced from cis plasmids containing a deletion in one of the two terminal resolution sites (trs). It can be provided to customers who have a research license to the technology. If you are interested in scAAV’s please provide proof of your research license with your order.
Because scAAV is double-stranded, its genome size is approximately half the size of that packaged in single-stranded AAV, corresponding to approximately 2 kb of foreign sequence.
Can you clone the AAV cis plasmid for me?
If you can provide us with the cDNA or expression cassette, we can clone the plasmid for you, confirm the identity by sequencing and restriction enzyme mapping, and produce AAV.
What quality control procedures do you use?
rAAV preps are subjected to the following procedures:
- Titer determination by quantitative PCR – usually performed on polyA sequences.
- Endotoxin assay.
- Purity assessment by SDS-PAGE.