Hi Talley

Both of AAV and lentivirus are suitable for shRNA expression. Your main consideration should be which virus could infect your target cell efficiently. Lentivirus is very suitable if you just want to infect primary neuron in vitro. Although the titer of AAV is very high, but AAV is good at infecting in vivo tissue but not in vitro cells. You may use AAV in hippocampus infection by Stereotactic injection.

We are very experienced in AAV and lentivirus production for in vivo or in vitro experiments. You could find more information on this website: www.genemedi.net/i/aav-packaging

thanks Melanie! that's all extremely helpful.

can you elaborate on the difficulty of purifying and concentrating AAV? given that you can get much higher titres with AAV, where does the difficulty arise? and in the case of in vitro stuff, where I might just be applying the viral supernatant directly (without concentrating... something I've had pretty good luck doing with lenti), do you think this consideration might be irrelevant?

also, do you have a preferred cloning system or vector plasmid for AAV RNAi?

thanks again.

Once again, thanks! I really appreciate the respone!

lentiviral vectors are largely better than rAAV because it's faster, cheaper to produce and purify these vectors are they are working pretty well. if you need info let me know

Imo there is no clearcut answer, which vector type is "better". It really depends on your goal.

E.g. we often express multiple proteins from one lentiviral vector (packaging capacity/max genome size 10.2 kb). AAV has 4.7 kb packaging capacity (ITR to ITR), just too small for multiple inserts and/ or dual-promoter constructs.

Lentivirus is an integrating vector. It may be used for lineage tracing, i.e. following transduced cells over many generations. As far as I know, AAV rarely integrates, and in its episomal form does not replicate along with the genome. So it will be lost over division cycles. @ Melanie (you're the expert): True...?

Lentivirus can be precisely targeted to certain cell types, using engineered envelopes. While AAV seroptypes show some degree of tropism, my impression is that their specificity cannot be tailored to the same extent as LV envelopes.

You don't care for any of these features - weell.... Lacking own experience with AAV, I have no opinion ;-)

Hi Talley! I think all of the comments from Melanie are very on point. I have used both types of viruses in vitro and in vivo and my feeling is that I always prefer to use AAV over Lenti if possible. The main issues for me are packaging limit and virus spread. Assuming you can fit your gene of interest into the AAV backbone (

Melanie mentioned in her first post that the time course of expression from AAV and lentiviruses is similar. I always thought that lentiviral protein expression could take up to a week or more in vivo. Does AAV also take that long, or is lenti actually faster than I thought? I am looking for a vector that will give rapid onset of expression and last about a week, in vivo, with minimal spread to surrounding tissues or across synapses. Any ideas would be appreciated.

@ Elisabeth:

Time course of LV expression in vivo and in vitro, two parameters:

- Time until you can detect expression (depends on your detection method)

- Time until expression has reached steady-state level

Pre-expression steps:

Both are slower than the "transient-transfection-of-naked-plasmid" scenario. AAV will not go through the (slow) reverse-transcription step of retro- (including lenti)viruses. But still the ssDNA genome needs to be converted into dsDNA, involving at least second-strand synthesis. Self-complementary AAV vectors (scAAV) have been made to sidestep in-vivo 2nd-strand synthesis, but are suitable only for small constructs (packaging capacity ITR to ITR: 4.7 kb for regular AAV, roughly half this size in scAAV. LTR to LTR in LV: 10.2 kb)

Integrated LV:

The single most influential factor on the onset of LV expression is the strength of your internal promoter, in the target cell type. For instance in neurons, lentiviral GFP driven by Synapsin promoter will be visible (using ordinary epifluorescence) ca. 30 h after transduction, steady state will be reached in ca. 48 h. GFP driven by CAG promoter, visible ca. 48 h, steady state ca. 4 days. So the rate of onset may be very different in two vectors of the same type (LV), depending on transcriptional efficiency.

AAV:

Serotypes differ dramatically in their ability to target various tissues and cell types. Choosing the right serotype for your target tissue will make the vector phenotypically "faster" because transduction efficiency is higher.

Spread:

Basically depends on two things, particle size and tissue density. Particle sizes: rAAV, 20 nm; LV, >100 nm. Smaller AAV particles will spread further (in a given tissue) than larger LV particles. Tissue density depends on brain region (e.g. directional spread in white matter), but most of all, on age. In embryonic to early-postnatal brains, LV will spread a lot (and AAV likewise, I suppose). In older animals, the difference may become more noticeable. In the adult, LV practically no longer spreads at all.

Duration:

In postmitotic cells, AAV expression is fairly stable. Colleagues have repeatedly imaged the same AAV-transduced cortical neurons in vivo, for weeks. In dividing cells, the AAV genome will not replicate along with the host genome, and expression will get lost depending on cellular growth rate (faster in fast-dividing cells).

I'm afraid there is no Jack-of-all-trades vector ;)

Alexandra - Thank you for your very informative answer.

Hi All,

I know this is quite late regarding the initial posting (about 30 months), but it would be great if some of you can comment on how benign or harmful AAV vs lentivirus are when injected intracerebrally and on for how long the RNA interference would be preserved. I need to knockdown some proteins followed by behavioral experiments that may last several weeks.

Thanks for any insights!!

I am, for the first time, using AAV carrying target gene to treat mice, could you please tell me what the optimal dose is for AAV in mice? Thank you!

AAV in vivo (4 years experience), there is no optimal dose, if you inject into the brain (?) like I did, then normally we used 10-25% dilution from the original stock, but this depends on your original titer of course. We normally got titers (thanks to our lovely AAV platform specialist) of 1E15 or 1E14, so we would give 1 ul aliquots of 1E13 or 1E14 of our strongest viruses into the brain.

Hi all,

I should start an experiment using stereotaxic adenovirus injection, but i don't have experience in this field. 

I need to inject an adenovirus cre recombinase in the dorsal raphe nuclei (5-HT cells) in flox/flox mice to knockdown an microRNA. Does someone know if there is an adenovirus suitable to do it? Any adenovirus cre recombinase is ok? it would be better if I can buy them "ready to use".

Thanks in advance

D

Hi, I am new to in vivo viral injection into embryonic brains. Can anyone advice on the dosage of AAV to inject? Also which AAV serotype produce more even neuronal expression in the forebrain cortex?  

Hi Lambert.

you already have got sufficient and great answers.

I just wanted to share our experience. LV have better transduction efficiency that AAV.

Why don't you try episomaly replicating viruses that would employ the host replication machinery for multiplication. Just a thought by the way!

There is a Lentivirus Webinar coming up that may able to help address your concern and any additional questions.

Title: Knockdown, Knockout, Validate: Lentivirus delivers payload in vitro and in vivo Date: Wednesday, May 16, 2018 Duration: 60 minutes Times: 10:00 AM (CST) Speaker: @Christy Hoffmann

What you will learn: Lentivirus biosafety features and best handling practices Flexible customization options to fit your needs Techniques to incorporate lentivirus into your gene studies Insight from a leading Lenti manufacturing expert

Register here: https://www.sigmaaldrich.com/life-science/learning-center/customer-education/lentivirus.html

Hi Melanie and Jonathan,

I'm planning to use virus to knockdown a gene in vivo (in the skin) of rodents. Have you ever worked with AAV or lentivirus for skin (probably via intradermal injection)? Companies recommended AAV type 2, but I've no idea how efficient and how fast will the shRNA delivery be. Thank you!

Hi Talley

Both of AAV and lentivirus are suitable for shRNA expression. Your main consideration should be which virus could infect your target cell efficiently. Lentivirus is very suitable if you just want to infect primary neuron in vitro. Although the titer of AAV is very high, but AAV is good at infecting in vivo tissue but not in vitro cells. You may use AAV in hippocampus infection by Stereotactic injection.

We are very experienced in AAV and lentivirus production for in vivo or in vitro experiments. You could find more information on this website: www.genemedi.net/i/aav-packaging