Dissertation Methods

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Dissection of Rat Brain

To peform dissection the equipment is sterilized with ethanol and rinsed with sterile wate before use and is perfomed under a laminar flow food.Rat Brain dissection was perfomed by my supervisor as I am yet to acquire enough experience. The brain is removed and placed into a petri dish with DMX. DMX is....DISSECTION MEDIUM - glucose (0.6%), penicillin/streptomycin (1x), hepes (10 mM) in PBS without Ca2+ or Mg2+. Keep at 4°C. Both hempispheres are transferred to a new dish after separation of the two cortices from the rat brain. Precision peeling of the dura matter is perfomed underneth a biocular microscope and Cortical tissues are transferre to a ne dish and cut into small pieces.

Cell Prep

Cortical cell preparation was completed in a sterile, class II, laminar flow hood with all equipment sterilied by IMS. Short pieces of cortical tissues are transfered to a tube for centrifugation, when settled the supernatant was removed and trypsin solution added. Tissues are digested for 10 minutes with swirling at 2 minute intervals. When the tissue is settled the supertant is removed and netralised by addition of 10ml horse serum and is left for 2 minutes with occasional swirling. This process is repeated with the removal of the supernatant and readdition of horse serum up to 10 ml. The suspended cell following dissasociation via a pippete is centrifuge at 300g (1399 rpm) for 8 minutes at room temperature. The supernatant is removed, resuspended in cell culture medium and is transfered through a 100um cell strainer.

Cell Plating.

Culture medium was used to increase the cell suspension and culture medium was increased to the prepared cell suspension so there is a cell suspension of 100,000-120,00 cells/ml. 200ul of cell suspension was pipetted onto each of the prepared coverslips. Plates are left in the incubator for an 1hr monitored for succesful plating. 2ml of cell culture medium was added.

Culture Media Changes.

Culture media half changes occur every 3-4 days. Viral transfection occured in the initial medium change and half media changes following.

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Cell Transfection Protocol and experimental groups.

Media changes are important as neuronal cells must be kept in specific conditions. Neuronal cell media should be changed every 3-4 days. Neurobasal medium is placed in room temperature before any cell application can take place.

Culture Media Protocol:

- Pen-strep x100= 1ml

- B27 x50= 0.5ml

-L glutamine x 200= 0.125ml

- Neurobasal media 48.375ml

Using a micropippete 1ml of Pen-Strep is added to 0.5ml of B27 and 0.125ml of L glutamine to the previously collect 48ml of neurobasal media. I usually gentle mix the solution following micropippeting. The culture medium is kept in the fridge if it is remaining following use. I must always ensure I label all of the medium.

Media Changes

For the different Cell groups to ensure that the cells are able to survive we must maintain conditions therefore it is important to change the media every 3 days to neuronal cells. Only half of the media is changed because a complete new batch of media would disturb the cell environment too much.

All work is of course performed under the hood

All of the ingredients must be sterilised with alcohol.

Nothing enter the hood without sterilisation

It is important when pipetting to only pipette until the first stop rather than all the way down otherwise measurements can be severely distorted.

When performing media changes, defer from pipetting centrally onto cell. Pipette on the side of the Tube so neuronal cells are no damaged.

Viral Transfection Protocol

Summary of Experimental goals and groups

As the goal of the experiment is to measure transfection efficiency in neuronal cells we have split cells into 3 main groups. day 2 , 6, 9. Furthermore we are transfecting the cells with 2 different AAV's. Syn GFP and CKII. Lastly we will use different quantities of AAV's for viral transfection 10^10 10^9 and 10^8

Insert AAV calculations image.

In viral transfection we remove 100ul /600ul and replace that with 100ul of the AAV dilution.

To Dilute to correct rations take the 10^10 dilute in culture media, take the dilution and dilute further into the 3rd test tube .

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Immunocytochemistry

Each of the five coverslips (1:200; 1:400; 1:600; 2'-only; and -ve control) should be imaged under UV light, blue light and finally green light. The slides are visualised under UV light to view the Hoechst stain, which stains cell bodies. They are visualised under blue light to view any green-staining due to goat anti-mouse Alexa-488 binding to the NeuN antibody; fluorescence under blue light thus shows the presence of neurons. Lastly, the cells should be visualised under green light to view any red-staining, which occurs as result of goat anti-rabbit Alexa fluor-546 binding to DARPP-32, which binds dopaminergic neurons/striatal neurons.

Based on the above, the slide to which no primary antibody was applied but secondary was (2'-only) should only have fluorescence when viewed under UV light. The negative control should not display any fluorescence under any light.

Images can be captured using Deltapix insight for further analysis/quantification.

Note: slides should be viewed in a dark room.

ELLIE STURMEY

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Key Descriptions

Adeno-associated viruses

AAVs have emerged as very powerful tools for gene delivery into neurons. Distinct capsid proteins expressed by different AAV serotypes result in the use of different cell surface receptors for cell entry and thus specific tropisms (Buning et al., 2008). Several of these AAV serotypes have been demonstrated to infect primary neurons with high efficiency and low toxicity (Royo et al., 2008), with AAV-2 being the most commonly used. Wild-type AAVs stably integrate into the human genome in a site-specific manner. Recombinant AAV-based vectors, however, integrate rarely and randomly because they lack the viral rep gene (Buning et al., 2008).

Since AAVs are naturally replication incompetent, they require a coinfection with an unrelated, wild-type helper virus (e.g., AdV, HSV) to supply essential gene products for the production of infectious virions. The new generation of recombinant AAVs is “helper-free” (while remaining replication deficient), eliminating the handling of an infectious, wild-type helper virus and simplifying the procedure. Moreover, by removing wild-type virus from the gene delivery procedure, the immune response of target cells is minimized. Limitations of AAVs are the late onset of transgene expression (∼2 weeks after infection), which hampers experiments with limited time frames, and a maximum insert size of ∼5 kb, restricting their use to smaller transgenes (Washbourne and McAllister, 2002).

AAV-Syn-GFP

AAV expression of GFP under the Syn promoterAAV expression of GFP under the Syn promoter