It sounds like your virus is no good (either expression vector or packaging). If you are doing stereotactic injections then the virus should transduce the target tissue as well as the cells along the injection path.  If you don't see this then the viral titeration is too low or there is something wrong with the viral prep.  There are no mysteries in molecular biology, and proper troubleshooting will answer your questions.

The only way ventricular targeting benefits you is if you are performing in utero injections (brain development 101) or if you really want to study the cells of the periventricular zone.

Hey Nathan, 

Depending on the viral titer, a miss into the ventricle should result in (random) transduction along the ventricular wall. Given the description of your problem, I tend to agree with Jason: sounds like a vital titer issue. But if you do see some periventricular  expression, a missed injection is a possibility. 

If your virus is AAV, you can test titer using qPCR or single MOI vital titration studies on a heterologous cell line (make sure the line actually expresses your promoter). Other than that, I'd check 1) production plasmid quality, and 2) whether the inter-ITR region of your transfer plasmid is < 4.2kb as higher won't package efficiently. 

If you're using other virus, troubleshooting steps are different, but similar. 

Best of luck, 

Joey 

We have often seen transduction in HC and ependymal tissues when animal at the upper limits of the weight range for particular stereotaxic coordinates receives intraventricular infusion instead of IT BLA, including viruses using an alpha-CamKII promoter. We have seen this with as little as 0.5uL containing around 5.0E11 viral particles, depending on the pseudotyped capsid protein. It sounds as though your titers may be low, in which case by refining your imaging conditions to eliminate extraneous light sources, and increasing your exposure time, you may begin to see faint paraventricular expression. Of note, we have created multiple AAV vectors that, at at comparable titers to other successful serotypes containing an identical insert, do not express in neurons in the region of the amygdala and EntCtX efficiently enough to reliably discern transduced cells from background fluorescence. These consistently include pseudtyped VP2 capsid sequences for AAV2, AAV4 and AAV5. They appear to require much higher titers and/or infusion volumes, which make them unsuitable for in vivo experiments using our purification protocols. This is a second possibility, but as your controls are expressing your reporter protein that's not necessarily a concern. Third, as has been pointed out, your insert may possess a flaw that hinders expression. Depending on the virus that you are using as your vector, if the insert exceeds the packaging limit you may experience inserts that are truncated at both ends. We believe that we may have experienced recombinant splicing of our insert consisting of the two truncated sequnces, forming a chimeric mRNA that bound to our oligonucleotides, giving a false positive, albeit low, result when we attempted to quantify using qPCR. Your controls show expression, so I would examine this carefully if it is a plausible scenario. I hope this helps you troubleshoot; knowing what vector you are using, and the protocols you are using to grow and purify the virus I might be able to share some of the insights we have gained using the processes we use.

Thank you all for your responses.  I should have specified that we are using an AAV5 vector that was made for us at the UNC Vector Core, property of the Diesseroth lab.  Therefore, the construct itself has been verified independently.  However, this leaves open the possibility of low titer for our specific batch.  I will definitely look to verify that.

I appreciate the advice!

You could inject  your viruses into superficial tissue (e.g. dorsal neocortex) where it is easier to hit your target (and avoid ventricles), and then verify the expression level of control and ChR2 variants. To reduce # of animals used, you could inject both hemispheres, each with one variant (but this runs the risk of misinterpreting potential retrograde transfection).

The Vector Core should tell you the titer for each virus, and you can then adjust the injection volume as needed.

What injection system are you using? The situation you describe could result from a clogged injection pipette, i.e. not actually delivering the volume that you think.

Thank you everyone for your advice.

As an update, I just got the histology on another animal in which I injected three distinct vectors in three regions, one being my vector that did not show expression previously.  I injected the problem vector into the sensory cortex this time, and there was still no expression.

I did get expression in the BLA with a different vector (same serotype, same promoter).

Therefore, I believe it is the vector itself that was ineffective, even though the reported titer was high (6 x 10^12 vg/mL).