Intracranial Injection of Adeno-associated Viral Vectors

Intracranial Injection of Adeno-associated Viral Vectors

Disinfectants at varying concentrations (hypochlorite: 5,000, 500, or 50 ppm; phenolic: 1:10 or 1:128 dilution; quaternary ammonium compound: 1:10 or 1:128 dilution) were added to either saline or whole blood (final concentration, 80% or 20% blood) and mixed. He had a history of urinary incontinence after undergoing surgery for prostate cancer 6 years earlier; his cancer was being treated with diethylstilbestrol. One such event was documented even as it happened in a three-day period. – May be used on food contact surfaces and as an instrument pre-cleaning spray or immersion solution. At 4306ppm dilution, BruTab6S kills: – C. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex. Ordering Information: Premoistened synthetic weave towels are available individually wrapped (21850-264); as a continuous, perforated roll in a canister (89042-154); or loose in a soft pack (47735-634).

These practices can be found in Biosafety in Microbiological and Biomedical Laboratories 5th edition, available on the CDC website(http://www.cdc.gov/od/OHS/biosfty/bmbl5/bmbl5toc.htm). The reef-mapping effort is part of a bigger project being carried out by the XL Caitlin Seaview Survey research team to capture thousands of reef images from all over the world. MMS also poses a significant health risk to consumers who may choose to use this product for self-treatment instead of seeking FDA-approved treatments for these conditions. All lenses showed positive HSV and adenovirus PCR results after inoculation and negative PCR results after washing with detergent/water and after disinfecting with bleach. Disinfecting hard, nonporous surfaces is one of the most reliable ways to help lower the risk of spreading these germs from surfaces by touch. Autoclave tape is not a fail-safe indicator of sterilization; it blackens after only brief exposure to a temperature of 121°C. 1 in 13 school-aged children has asthma.

Dispose of plastic waste in a biohazard container as instructed by your institute’s biosafety officer. Cover the surgical area with absorbent lab bench paper. Surgical tools should be sterilized and surgery done under aseptic conditions in accordance with your institution’s biosafety and animal use guidelines. Choose an area adjacent to the surgical area that will be dedicated to loading the micropipettes with virus, and cover it with absorbent lab bench paper. Place an aliquot of virus in a container of ice in this area, and allow the virus to thaw on ice as the surgery is being performed. Viruses are abundant, normal and diverse residents of stony coral colonies, the researchers noted in their study. Rice has highly respected schools of Architecture, Business, Continuing Studies, Engineering, Humanities, Music, Natural Sciences and Social Sciences and is home to the Baker Institute for Public Policy.

Inject a mouse subcutaneously with buprenorphine at a dose of 0.5 mg/kg. H, Medical Director and Sally G, M.S.W. Place the tip of the micropipette into the viral stock and manually withdraw the plunger. If difficulty is experienced drawing viral stock into the micropipette, the tip can be enlarged slightly be piercing a KimWipe with the micropipette to break a small portion of the glass. Lower micropump arm onto plunger until a small amount of virus is dispelled from the tip of the micropipette. Remove this small drop with a cotton tip applicator and discard applicator in waste container. Using X and Y stereotaxic coordinates, position the micropipette over the area to be injected.

Intracranial Injection of Adeno-associated Viral Vectors
In this experiment, the stereotaxic coordinates used to locate primary visual cortex are 2.7mm posterior to bregma and 2.5mm lateral to the midline. Very slowly lower the micropipette (at an approximate rate of 1mm/1minute) to the proper Z position. Enter the desired injection parameters into the SYS Micro4 micropump controller box and initiate the injection. For this experiment, 1 microliter over 10 minutes will be used as a rate of injection. When the injection is finished, leave the pipette to rest for one-two minutes to prevent efflux of virus during removal. Is it ok to overlap MMS with current medication? Suture the scalp and seal it with tissue glue.

Inject the animal subcutaneously with buprenorphine at a dose of 0.1 mg/kg every 8-12 hours over the next 72 hours, or as long as the animal is exhibiting signs of pain. Surface compatibility-bleach will corrode many metals, rinse with water after use; instruments vary in their ability to withstand disinfectants based on their composition. Imaging experiments can be initiated after the desired incubation time (days to weeks after viral injection). Figure 1. Transduced neuron after injection of double-stranded adeno-associated virus serotype 1 (dsAAV S1) carrying green fluorescent protein (GFP) under control of the CMV promotor. The cell body as well as proximal and distal dendrites are clearly visible in fixed sections imaged using an epifluorescent microscope. Scale bar= 100 μm.

Figure 2. Labeling typical of an intracranial virus injection in primary visual cortex using dsAAV S1 showing the extent of viral spread as well as labeled neurons, glia and processes. Scale bar= 250 μm. Virally-mediated gene delivery holds great potential for the study of neurological processes and treatment of brain disorders1,2,3. Renji Abarai – Bleach Wiki – Your guide to the Bleach… Here we demonstrate a detailed procedure for the transduction of neurons and glia in mouse visual cortex using a double-stranded adeno-association virus expressing enhanced green fluorescent protein. While this technique is relatively straight forward, there are a number of technical details that need to be considered.

One important factor is injection-induced tissue damage – therefore it is crucial to use great caution during the surgery and insertion/removal of the micropipette. A failed surgery could result in the alteration of the structures to be imaged. Additionally, after loading the virus into the micropipette, great care must be taken to clear any air bubbles at the pipette tip and ensure the proper volume is injected. Lowering the micropipette slightly beyond the target Z-coordinate then withdrawing it to the proper position can prevent virus from overflowing out of the injection site. When choosing an incubation time, allowing adequate time for viral gene expression should be considered. This will vary based on the virus used. A limited immune response may be elicited by the injection.

In our experience, this inflammation is generally restricted to the needle track and can be avoided by imaging away from the injection sight (approximately 50 μms or more). This is not a case of a supplement adulterated with a prescription drug or containing trace amounts of heavy metals. Intracranial injection of viral vectors has several technical advantages over other labeling techniques. Through the use of stereotaxic coordinates and modulating the volume injected, fluorescent label can be precisely localized to an area of interest. For example, E614293 = production date 10/20/2014 (October 20th is the 293rd day of 2014). Additionally, the use of different viruses (e.g. lentivirus, herpes virus, adenovirus) can also modulate the timing and amount of fluorescent protein expressed as well as the cell types targeted.

Overall, this technique can provide for very versatile labeling of different brain elements for imaging. The animal and experimental protocols were approved by the University of Rochester University Committee on Animal Resources (UCAR) in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals. This work was made possible by grants from the NIH (EY012977), a Career Award in the Biomedical Sciences from the Burroughs Wellcome Fund, the Whitehall Foundation, and the Sloan Foundation (A.K.M.).

You may also like