Adeno-Associated-Virus ( AAV ) FAQs

1. How can I choose the optimal AAV serotypes for my in vivo study?

Genemedi has launched a comprehensive AAV production service. More than 12 AAV serotypes and a variety of capsid engineered AAV vectors are available for targeting different tissues and organs. Different AAV serotypes have different tissue tropism in vivo, the common serotypes and their tropism are listed in the above table 1. Therefore, you can select the most suitable AAV serotype for your study according to this table. For example, if you’d like to transfect your target gene into mouse ears, the AAV-Anc80 will be your best choice.

2. How much DNA do I need to provide for Custom AAV without DNA amplification?

You will need to provide purified plasmid DNA at a concentration of 0.5ug/ul or more.

50 ug DNA needed for Custom AAV production service without DNA amplification (10^9 GC/ml)

200 ug DNA needed for Custom AAV production service without DNA amplification (10^12 GC/ml)

300 ug DNA needed for Custom AAV production service without DNA amplification (10^13 GC/ml).

3.What's the difference of AAV titer unit between GC/ml and vg/ml?

The AAV titer unit GC/ml and vg/ml can be used interchangeably, based on qPCR method.

4. Why can't I see the expression of GFP after AAV infection in my cell line?

AAV serotypes selection is an important parameter which may affect the transduction ability of AAV particles. Thus, it is necessary to determine which serotype works best for your cell line. For instance, serotype 5 limits its transduction ability on most cell types. For detailed information, you can refer to our technical sheet "AAV - General Guideline to Serotype Selection".

5. Does Genemedi's AAV preparations belong to in-vivo grade?

Extensive purification steps guarantee the high quality of our viral particles, which are ready to be administrated for in-vivo models. For detailed references, please see the in-vivo infection data tested by another independent lab { www.genemedi.net }

6. What is the QC (quality control) method for testing AAV in Genemedi?

We provide qPCR-based titer as the primary method to determine whether the packaging/purification process is successful or not. If your virus has GFP or RFP reporter, we also perform virus infectivity testing in HEK293T cell line.

7. Can I utilize different serotypes of AAV virus in the same equipment to keep the infected cells?

There is no problem with using different serotypes in the same equipment, as long as the handler takes the basic precautions to avoid cross-contamination.

8. What is the difference between brain localization of gene expression after injection of AAV or lentiviral vectors?

Lentiviral particles don't spread well after stereotaxic injection into brain because the particles are relatively large (90nm~100nm). In contrast, AAV particles spread more readily due to smaller size (20nm~100nm). As mentioned, it has been stated that some AAV serotypes spread better than others in brain, for example, AAV5 is reported to spread exceptionally well when injected into striatum.  Lastly, pretreatment with mannitol to your animal about 15 min ahead of viral injection has been reported to aid in spread of viral particles in the brain.

9. Do AAVs tranduce axons passing through a region of interest?

AAVs are able to infect axon terminals and produce retrograde transport (towards the cell body). The slightly longer answer is that this process is highly biased based on the serotype you are using. There are a number of papers reporting AAV6 (and maybe AAV6.2) is the main serotype to produce retrograde transport. There are however reports of many other serotypes producing retrograde transport with a much smaller rate.

10. How can I increase the transduction efficiency?

Try varying particle-to-cell ratio (PPC), incubation volume, temperature and, cell density (if adherent cells are transduced). For adherent cells, we recommend a confluence of about 70%. Following the PPC, adjusting the volume is the next best parameter to change to optimize protein expression.

11. What is the difference between LC3A, LC3B and LC3C, or LC3-I and LC3-II?

LC3 is a soluble protein with a molecular mass of ∼17 kD and is distributed ubiquitously in eukaryotes. It is expressed as the splice variants LC3A, LC3B, and LC3C which display unique tissue distribution. All LC3 isoforms undergo post-translational modifications, especially PE conjugation (lipidation) during autophagy. Upon autophagic signal, the cytosolic form of LC3 (LC3-I) is conjugated to PE to form LC3-PE conjugate (LC3-II), which is recruited to the autophagosome membranes.

12. Will the GFP signal of mRFP-GFP-LC3 that anchored at autophagosomes and fused with lysosome relight after the cell fixed?

GFP is no longer delighted reversibly once GFP-LC3 signal was destroyed by the acidic environment.

13. Can transient expression of RFP-GFP-LC3 differ badly from stable expression?

Transient transfection of GFP-LC3 leads to the formation of non-autophagic LC3 puncta due to overexpression. Reports shows that GFP-LC3 is aggregate prone protein. We suggest the use of stable cell line for assessment of GFP-LC3 puncta.

14. Which software do you usually use for counting the number of LC3 dots in the cell?

Image J does work. For LC3 puncta, we suggest using the "Watershed" plugin from Image J. But proper threshold adjustment is critical. CellProfiler is another choice. First use the top hat filter. Then with the Identify Primary Objects module, manually adjust the Intensity threshold to optimally select the punctas of the right intensity.

15. How long is half-life time of chloroquine when I treat a cell line for autophagy study?

Chloroquine is an attractive drug agent effective for the treatment of not only malaria but also inhibition of autophagy, which is a promising effect for anti-tumor therapy. Half-life time of this drug is approximately 18 hours in vivo due to the degradation by the liver, but the stability of chloroquine in vitro experiments is expected to be longer. It will depend mainly on the cell line you are working on and their metabolic activity. For example, HepG2 cells (human hepatocarcinoma cells) have a strong capacity to metabolize drugs, which might not be the case for other cell line of different tissular origin. I would suggest to add chloroquine when you change your growth medium.

16. Which medium is best to use to induce LC3 puncta in HeLa cells?

We recommend the media used by Axe et al. (2008) JCB 182: 685-701. The recipe is 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 20 mM Hepes, pH 7.4, Add 1% BSA and pass through a 20um filter before use.

17. What is the best applicable inhibitor of autophagy?

I prefer to use CQ (chloroquine) in all of my researches about autophagy inhibition. The using of CQ is quite easy, since it is easy dissolve in water (unlike 3-MA in DMSO). One time I have been used 3-MA, actually I did not like to work with it. Bafilomycin is another choice for you.

18. When to add bafilomycin to study autophagy?

It is better to do a time course to make sure. We use at least two times (2 and 4 hours) to starve the cells incubate bafilomycin for 2 hours (for 4 hours: the 2 last hours). Then LC3-II and P62 are detected by WB. For the degradation of p62, sometimes, 4 hours is better than 2 hours.

19. Which neuronal cell line should I use for autophagy level?

SHSY5Y and PC12 are two well established neuronal cell lines, human and rat cell lines, respectively. You may also try Neuro2A, it is a mouse cell line.

20. How to distinguish selective and non-selective autophagy?

You can analyze selective mitophagy by interaction/co-localization study of mitochondrial marker protein and the autophagy specific adaptor protein LC3II.

21. When to add the autophagy inducers or inhibitors in cell culture models?

First, some cells are very sensitive to these inhibitors and you need to optimize your conditions. The second point, if you like to stop autophagy process after inducing it to compare whether it is a cell death or cell survival you may add it just hours (2-3) before treatment. However, many articles prefer to use it as pre-treatment 1 hour before any other treatment.

22. Is it still possible to get the same mRNA accumulation in RT PCR after CRISPR/Cas9 induced gene deletion?

Yes. The transcript will continue to be transcribed as nothing has changed with respect to the promoter of the gene. Besides the indel you introduced, the transcript will be unchanged and your primers (presumably not overlapping indel) will detect the transcript. You'll want to use Western blot to observe the effect of the indel.

23. I am trying to design gRNA for TLR4 gene CRISPR cas9 genome editing. TLR4 has multiple transcript variants. Which one should I choose?

You should use a gRNA which target as many transcript variants as possible. To make it clear, target those exon(s) which can be found in the most variants of TLR4. As I see exon 1 and exon 4 are the best choices to target TLR4.

24. Which sequencing depth should I use for my CRISPR-Cas9 screen?

PCR amplification and Sanger sequencing are the main CRISPR KO screening methods.

25. Does the CRISPR/Cas9 system work for non-sequenced organisms?

In theory it should be able to work with any organism. However, there may be some modifications that you will need to work out. You may need to codon optimize the Cas9 gene for your organism and confirm if there are many rare codons used in your organism. Next, you'll need to find a good promoter in your organism that you can use to make your crRNA. Maybe you'll need to add a nucleus localization signal to the Cas9 to make sure it stays in the nucleus, for Cas9 won't work if it is in the cytoplasm. Another issue you need to determine is what protospacer to use. If your organism is similar to one that is sequenced, then this may work, otherwise if you don't know the DNA sequence of your gene, it will be impossible to design a good protospacer against it. Lastly, you'll need a way to transfer Cas9 and crRNA into your organism, which should not hurt your organism.

26. What's the simplest and most efficient optogenetics setup for dual (independent) light delivery, such as blue for ChR2 and green for eArch3.0?

A blue LED coupled to a fiber for ChR2 and a green laser with optical shutter for Arch3 can be recommended. LEDs are easier to drive, but a laser is probably required for inhibitory ion pumps.

1) LEDs have nearly linear electrical current to optical power output, so if your study requires highly precise temporal patterns of stimulation, this would be the way to go. However, LEDs emit light in many directions, so it is difficult to focus the light into the fiber. The ability to couple a high percentage of the LED's output into a small diameter fiber, the coupling efficiency, is a key way to differentiate the available products.

Lasers can be coupled at a much higher efficiency. However, cheap diode lasers perform quite poorly when pulsed. A laser rated at 100 mW might only put out 3 mW when pulse for 1 ms. The best way to use a cheap dpss laser is to leave the laser on and then gate the light with optical shutter. However, this approach can only be used for rectangular pulses, not arbitrary temporal patterns.

2) Miniaturized LEDs help get more light into the end of the fiber.

3) If you can get >5 mW out of a 200 μm fiber, you'll have more than enough for the ChR2 studies, but inhibitory ion pumps require relatively more light. A green laser with an optical shutter would probably be best.

AAV Rep-Cap plasmids (serotypes-specific AAV RC plasmids)