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Viral Vectors 101: Parts of the AAV Packaging Plasmid

Posted by Beth Kenkel on Dec 3, 2020 9:15:00 AM

Your next cool experiment requires some AAV.  Where do you start? Plasmids of course! You just need three plasmids to start making the AAVs you need for your experiment: 

  1. the packaging plasmid which contains the AAV structural and packaging genes,
  2. the adenoviral helper plasmid which contains the proteins needed for the virus to replicate, 
  3. and the transfer plasmid which contains the viral genome.

In today’s blog post, we’ll focus on the packaging plasmid and take a look at each of its parts.

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Topics: Viral Vectors, Viral Vectors 101, AAV

Channelrhodopsins with improved light sensitivity for minimally-invasive optogenetics

Posted by Beth Kenkel on Nov 3, 2020 9:15:00 AM

Optogenetics is a neuroscience method that lets you fire neurons with the flick of a light switch. Neurons are not typically persuaded to fire when light is shined on them, but the expression of light-gated ion channels such as channelrhodopsins (ChRs) makes them light-responsive. When light shines on excitatory channelrhodopsin-expressing neurons, the channelrhodopsins respond by opening and allowing an influx of ions into the neuron which generates an action potential (Figure 1). This light-induced excitation can take place in a tissue culture dish but can also happen in real-time inside a mouse’s brain. 

But the brain is a tricky organ to access, which often forces scientists to use invasive measures to perform in vivo optogenetic experiments. Intracranial injections of AAV are commonly used to deliver channelrhodopsins to the brain. This delivery route concentrates lots of channelrhodopsins in one region of the brain, which is needed since current channelrhodopsins tools have low conductance, but also confines channelrhodopsin activation to a small volume of brain tissue (~1 mm3). Current channelrhodopsins also need high-intensity light for activation which requires implantation of fiber-optic cables into the brain.

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Topics: Optogenetics, Viral Vectors, Neuroscience

Mycoplasma Contamination: Where Does It Come From and How to Prevent It

Posted by Guest Blogger on Oct 8, 2020 9:15:00 AM

This post was contributed by guest blogger, Kaustubh Kishor Jadhav, a Research Assistant at MGMs Institute of Biosciences and Technology.

If you are reading this article then you probably suspect mycoplasma contamination in your cell culture or you are about to begin a new cell culture project. If mycoplasmas are present in your lab, don’t be surprised. They are present in most of the cell culture facilities, tissue culture labs and every cell culturist has to deal with this problem. It is estimated that mycoplasma is responsible for up to 60% of the cell culture contamination (Uphoff, 2002).

Mycoplasmas are considered to be one of the simplest and smallest bacteria. The absence of a rigid cell wall makes them resistant to antibiotics and antibacterial drugs like penicillin and streptomycin. Mycoplasma can pass through filtration methods because of its ability to change shape and the absence of a rigid cell wall. Here, I will cover some of the best ways to tackle mycoplasma contamination before they enter your cell culture and what to do if you encounter mycoplasma contamination.

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Topics: Viral Vectors, Viral Vector Protocols and Tips

Four Ways to Package Transgenes That Exceed the Size Limit of Adeno-associated Virus

Posted by Beth Kenkel on Sep 1, 2020 9:15:00 AM

Adeno-associated virus (AAV) has many features which make it a great viral vector, but its packaging capacity is limited to ~4.7kb, or roughly half the packaging limits of lentiviral and adenoviral vectors. While many transgene will fit within this limit, some like prime editing's PE2 enzyme do not. So how do you fit a big gene into a tiny vector like AAV? By breaking the transgene into smaller pieces.

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Topics: Viral Vectors, AAV

Viral Vectors 101: Parts of the AAV Transfer Plasmid

Posted by Beth Kenkel on Aug 11, 2020 9:15:00 AM

So you have this awesome experiment you want to do, but it requires some AAV. You’ve never worked with AAV before, but you aren’t going to let that stop you. Where do you start? Turns out like all good experiments, making AAV starts with some plasmids. You just need three plasmids to start making AAV

  1. the packaging plasmid which contains the structural and packaging genes,
  2. the adenoviral helper plasmid which contains the proteins needed for the virus to replicate, 
  3. and the transfer plasmid which contains the viral genome.

In today’s blog post, we’ll focus on the AAV transfer plasmid and take a look at each of its parts.

Read More >

Topics: Viral Vectors, Viral Vectors 101, AAV

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