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1. Why does adenovirus have a relatively higher immunogenecity compared with rAAV? 

The adenovirus without E1/E3 can express all of the other genes in the viral backbone and hence induces immunogenic responses, while rAAV does not have any of the AAV genes, thus no immunogenicity from viral protein.

2. What is the role of the E1 Gene in adenoviruses?

Simply, the E1 gene products are early proteins that are transcribed in the early transcribed regions and required for proceeding subsequent steps in viral replication. The E1 gene contains E1A and E1B, involved in the replication of adenovirus. E1A is critical to start viral replication by promoting transcription from rep gene promoters, P5 and P19, and facilitate viral replication by activating the early adenovirus promoters. 

3. How to store adenovirus?

It would be better to store adenovirus in PBS at -80oC. Sucrose or DMSO may help to stabilize the vector. 

4. How can you tell if your vector is lentiviral, retroviral, or adenoviral? 

You should blast your vector sequence and see if there’re sequences of reverse transcriptase and integrase (gene names: gag and pol), which are for lentiviral/retroviral vectors, but not for adenoviral. For another, if your plasmid is around 30-35kb in size, it's certainly adenoviral.

5. Is adenovirus a useful tool to study primary macrophage functions?

In RAW264.7 and PM cells, adenovirus works very well, and it seems that IL1β expression is increased slightly after adenovirus transfection compared with negative control. While for the BMDM, adenovirus does not work, it may be better to use lentivirus instead, which gives a pretty good transduction efficacy and less inflammatory response. 

6. Is it possible to infect a tissue preparation with lentivirus and afterwards with adenovirus and getting high efficiency in transduction?

It is definitively possible to perform sequential transductions/infections. Polybreen, protamine sulfate or other transduction enhancing reagents are recommended to enhance viral particle infectivity.

7. How to perform adenovirus transduction in vitro?

First, amplify to get enough target cells, then infect the target cells with adenovirus. Cell functional assay can be conducted after expression of the target gene. Generally, infected cells will not be amplified for culture.

8. 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 kDa 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 autophagosomal membranes.

9. Can I use the Ad-mRFP-GFP-LC3 products to study autophagy in cells in combination with a lentivirus transfection?

Adenovirus transfection may actually slightly induce autophagy, skewing the results. Make certain to include appropriate controls and make sure to leave an appropriate time (48-72hrs) post transfection to lower the basal autophagy level.

10. 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.

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

Transient transfection of GFP-LC3 can 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.

12. 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.

13. How long is chloroquine half life when I treat a cell line for an 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 your are working on and their metabolic activity. For example HepG2 cells (human hepatocarcinoma cells) have a strong capacity to metabolize drugs. this 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

14. What 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.

15. 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.

16. When to add bafilomycin to study autophagy?

it is better to do a time course to be sure. We use at least two times (2 and 4 hours) to starved the cells and we add baf for 2 hours (for 4 hours: the 2 last hours). We then follow LC3-II and P62 by WB. To see the degradation of p62, sommetime 4 hours is better than 2hours.

17. 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.

18. 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.

19. When to add the autophagy treament 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. 

20. 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.

21. 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.

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

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

23. 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.

24. Why is spCas9 the dominant CRISPR system?

One of the main reasons is the relatively abundant PAM (protospacer adjacent motif) sequence of spCas9: NGG. This sequence is highly abundant throughout the genome, so this will give you a large amount of options for the sequence to target your sgRNA against. For instance, ST1 and NM require PAMs NNAGAAW and NNNNGATT respectively. These are far less abundant, and thus give far less targets in the genome to work with. SaCas9 is indeed a lot smaller than spCas9, and this can be Advantageous. However, its optimal PAM is NNGRRT. 

The second is that SpCas9 outperforms Sa/Nm in generating DSBs in the genome.

The third part of the reason is also for historical reasons. There may be Cas9 species with other 3nt PAM sites. However, since the first one that was discovered was SpCas9, most systems have made adaptations of this Cas9 protein, so sgRNAs (and sgRNA design tools) can be used for multiple systems (including dCas9 activation / inhibition).