Hello, my name is Ian Snapp, and this is the fourth week of my neuroscience internship at the Scripps DNC. To recap, I am working under the Ye lab, studying metabolism within the nervous system. This research requires a focus on an incredibly minute scale, involving molecular biology and biochemistry manipulating DNA to change metabolic patterns inside of neurons.

Things I Have Learned

Throughout this week, I was able to piece together some more information on the latter stages of how the DNA alterations are actually analyzed. As aforementioned in my previous posts, most of the work is determining the DNA variable of change, changing that variable, and trying to get it into a neuron. What I learned was that once we have the virus, aka, the vehicle to move the DNA, we infect only a colony of cancer neuron-like cells. I previously learned that this is because they are cheap, rapidly reproducible, and close enough to mimic the reaction of a neuron. However, what I discovered was that this is to test if there is a positive result or some recognizable pattern from our DNA transfection. If so, the focus shifts towards infecting neurons inside of a living mouse. Meaning the cancer cells are just an intermediate stepping stone to determine any change, and the real study is in primary cells and living mouse cells (in vivo).

The cells in the primary culture do not exhibit any cell genesis, reproduction, which makes them expensive and hard to obtain. However, these cells, typically extracted from a baby mouse brain, are nearly identical to human neurons creating the most accurate and detailed experiment to determine the effects of wherever manipulations inside the DNA have occurred. Infecting mice, on the other hand, allows for behavioral observations to be made and a better idea of how the DNA expresses itself in a complete network of neurons inside of a brain. Once the behavior studies have been completed, the mouse is killed, dissected, and the brain can be imaged to visualize the regional effects of the DNA manipulation.

Monday

To start off the week, we did a lab meeting downstairs in the auditorium (as usual). Once the meeting had finished, Dawn and I sent off the DNA we extracted Friday for sequencing to determine if our procedures had worked up to this point. While we had a bit of downtime, we ran an autoclave cycle to sterilize our dissection tools so we can extract neuronal tissue from mice pups tomorrow. After a quick lunch break, I watched as Dawn split HEK cells. Once Dawn finished up with the TC bench, Leyao transfected another culture of HEK cells with our AAV virus (both the DNA to construct the capsid body and the sample DNA). To finish off the day, Leyao prepared dishes for primary cells (neurons) for tomorrow. This essentially means binding a polymer to the base glass of the plate which the neurons can attach to, removing them from suspension inside of the solution, and allowing us to image them down the road.

Leyao is prepping the bench to add the polymers to the plates for the primary cells tomorrow.

Tuesday

On Tuesday, Leyao dissected mice pups and isolated cortical neurons (neurons from the cortex region of the brain). These neurons were then suspended into a buffer solution and transferred into the previously prepared plates. After that, I watched as Dawn transfected more viral solutions. To finish off the day, I made a batch of agar plates which are essentially a piece of gel used to help foster microbial growth.

This is Leyao using a multichannel pipette to inject the cortical neurons (primary cells) into the plate wells prepped on Monday. Don’t worry, the red color is the buffer solution, it isn’t blood.

Wednesday

Wednesday was a short day for me. All I really did was watch a perfusion, which means killing a mouse that has had the DNA expressed in its brain, flushing its circulatory system out with chemicals, and cutting the brain out. Once that was finished, I made LB broth (water and a nutritious medium) and added bacteria into a large flask of LB to prepare for a Midiprep on Thursday.

I am prepping for a Midiprep by adding a frozen bacteria sample, containing the sample DNA, into a larger flask full of LB broth to rapidly produce a large bacteria colony overnight.

Thursday

Thursday morning, I did a midiprep with the bacteria solutions (containing four DNA samples). Unfortunately, one flask broke, destroying one sample, and a filter broke, ruining another, so I only finished with two. After the rest of the lab finished their lunch, I watched Leyao collect the HEK cells and supernatant solution (the liquid the HEK cells were suspended in) to extract the virus later. The last thing I did was prep another bacteria solution to replace the two missing from earlier. This entailed transfecting more bacteria with our sample DNA and putting it on agar plates for later.

Aside from work, my mom flew out to see me for the weekend, arriving in the afternoon. Once I picked her up, we went back to the rental and just spent time talking for most of the afternoon.

These are the column filters used to remove most of the debris and liquid from the solution after the bacteria is lysed (destroyed freeing the DNA).

Friday

To finish up the week, I ran a much-needed load of dishes, went to a seminar, and watched Dawn harvest some cells. Additionally, I was able to see her cut thin slices of the mouse brain extracted Wednesday for imaging later. This was done by freezing the brain in a gel, and thinly cutting it with a razor edge all with a large fancy machine that cools the tools (to prevent the gel from thawing) and provides the right angle for cutting.

Once I left work, my mom and I hung out again, going to Mission Beach and then to a sushi restaurant. When we arrived back at the rental, we talked again, watched a movie, and planned out a zoo trip for Saturday.

The thin slices of brain tissue suspended in a solution until we image them next week.

Saturday

To finish off the week, my mom and I went out on our zoo trip, spending all day walking around the park. Eight-plus miles later, we had seen everything we could and decided to leave to go get sushi before going back to the rental.

A couple of the giraffes we saw at the San Diego Zoo.

My mom will be leaving later Sunday, but we plan to go back to Balboa Park to explore more of the area on foot before I drive her back to the airport. I am grateful she took the time to come hang out and explore San Diego with me, it really made the trip that much more enjoyable.