doc w/ pen

a journalist becomes a doctor before your eyes

Category: Lab Work

It’s All About the Balls

Female mouse pup –
completely smooth underneath.

I survived Week 1 of my supervisor being on vacation. My excitement for the week: learning how to definitively tell mouse pup females from males. I know what you’re probably thinking: boys have a penis, girls don’t. How hard could it be? Well, these little pups are so tiny (much smaller than a kiwi, as a point of reference), and probably weigh 10 – 15 grams. Itsy bitsy. The outward anatomy is sometimes hard to distinguish. (And I’m not the only one who has difficulty with this – I was looking at a sick mouse with the animal facility veterinarian on Friday morning, and she thought a male pup was a female at first! So I don’t feel so bad.)

Male pup – visible testicles.

I was splitting cages Friday afternoon (separating pups from their parents, males pups in one cage and females in another cage). From the outward genitalia, some were easy to tell, some not so much. Then I realized: the females are completely flat underneath, while if you hold the males long enough and let them squirm a bit, their testicles bulge out. Bingo! Definitive sexing. And my supervisor will be quite happy that I got the right mice in the right cages, because if you don’t, the males quickly impregnate the females.

It’s the little things, right?

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Library Access: A Lab Job Perk

PubMed is a great resource. For those of you who aren’t familiar with it, PubMed is an online resource hosted by the U.S. National Library of Medicine and the NIH. It has more than 22 million citations to biomedical literature. Some of those citations have links to full text articles. But unfortunately, many of the more prestigious journals charge for their articles (often as much as $30 or $40 per article). Given my current broke status, I can’t exactly afford to buy article access.

So I recently e-mailed my PI at the UIC lab where I work asking whether I could get journal access through UIC (major universities usually provide this type of access to their students and some employees). He told me that all I needed to do was plug my laptop into the UIC network (via a network cable at the lab) and I would automatically get access to everything.

A few weeks ago, I spent a little downtime getting some of the articles I wanted. When you download an article from some of these publishers’ sites, a little window will pop up with “recommended readings” based on what you just looked up. I noticed at some point that not a journal article, but a book chapter, popped up as one of those recommended readings. I hadn’t thought about book chapters. But as I did think about it, the idea appealed to me. A book chapter would provide a great deal of background, which is what I am looking for right now. So I wondered, “Could I get the whole book?” The answer: “Yes!” Then my follow-up question: “Are there more available books on my subject?” The answer, again: “Yes!”

From home, I used the UIC library’s Web site to find available eBooks. Then while a gel was running at lab, I downloaded them, chapter by chapter (you can’t download the whole book directly). Back at home, I put them on my iPad.

Granted, I’m not going to read every single page of every single book I downloaded any time soon. But I want to be able to skim them, to decide what I want to read and not read. So getting entire books made sense.

After I had finished getting my books, I was curious – how much would all of these books have cost had I purchased them? Being the absolutely nerdy person I am, I made an Excel spreadsheet to calculate the cost of buying the hardback book directly from the publisher, the eBook from the publisher, the hardback from Amazon.com, and the eBook from Amazon.com (yes, I actually did this). I was amazed – the hardback books from the publishers would have cost more than $2,000 (eBooks and Amazon.com hardback prices were somewhat less). One more reason to be affiliated with a major research institution, and to be grateful for my lab job!

Here is my lovely spreadsheet:

library savings

New [Mouse] Surgeon on the Block

Until a couple of weeks ago, I had mainly been doing genotyping at my lab job. I’ve got it down to a “science” (pun intended). I’ve been getting great results, which is wonderful. But I was itching to learn some new techniques. Well, I’ve gotten my wish.

My supervisor is teaching me animal surgeries. She has dozens of these to perform in the coming weeks and months, and wants someone to help reduce her load. And of course, I’m thrilled to learn something so practical for my future career as a physician-scientist, especially given that the mouse is the most frequently used animal model for diabetes research, which is what I want to do.

The first technique I learned (and am now pretty good at) is called an ELW (Excess Lung Water) procedure. It involves nebulizing mice with LPS, which basically gives the mice a septic lung infection, and then measuring various aspects of their lungs and blood. The most difficult part is taking a blood sample from the inferior vena cava, which you can imagine is pretty tiny in a mouse. And given that I’ve never really handled a syringe before, getting that needle in and then pulling the plunger back (with the same hand) was at first a challenge. But Thursday I performed my first ELWs on experimental, as opposed to practice, mice, and all went relatively well. (Except for one thing, which I will talk about in another post.)

The other procedure I’m learning is much tricker, and I’ve only mastered the first half. The purpose of it is to clear the mouse’s lungs of blood so they can be used for other experiments, such as histology, sectioning, etc. Cutting out the lungs is the easy part. The more difficult parts are putting the mouse on a ventilator (yep) and catheterizing the heart. Getting the mouse ventilated involves cutting part way through the trachea (again, quite small in a mouse), inserting a trach tube, and then hooking that up to a ventilator machine. The hard part is all the manipulations you have to do with your forceps prior to getting the trach tube in – for example, getting the 90-degree forceps under the trachea without causing the mouse to go into tracheal spasms (so you can pull through silk thread to eventually secure the trach tube). At first, I really struggled with getting that trach tube in. But I am quite good at it now, which is very exciting progress for me.

After getting the mouse on the ventilator, I heparinize the mouse to prevent blood clots, again through the IVC. Not that bad, considering I now am pretty decent with the ELWs. Another challenge after heparinization is catheterizing the heart. A mouse’s heart is literally the size of my pinky fingernail (and I have small hands). You have to get silk thread under the pulmonary artery, cut off half of the atrium, cut the aorta/IVC, and then slice slightly into the left ventricle to insert a small catheter. You then feed the catheter up the heart, into the pulmonary artery, so the fluid (PBS) going through the catheter will clear the lungs. This part of the procedure I am not so good with yet, but I am making progress. “Paso a paso,” one step at a time.

It’s slightly amusing to me that I am doing these procedures, and really enjoying learning them, given my past history with animal dissections. When I was a kid in homeschool, my mom would go to the butcher and get meat remnants (eyeballs, a pig head, various organs) for us to dissect. My sister would totally go to town with them, using a surgical kit that my dad, a physician, lent us. I wasn’t afraid of the dissections, but neither was I interested in them, so I hung back, watching. Now I’m totally into it, and thrilled to be expanding my skill set, as well as becoming more useful in the lab.

Long-Overdue Update

Well, it seems it’s been 2 months since I last posted. How time flies, doesn’t it? But I’ve been keeping myself out of trouble, I promise. Here is a bit about what I’ve been up to …

I wrote in a previous post that I had a tentative part-time job at the University of Illinois-Chicago Anesthesiology lab where I worked as an unpaid assistant for two summers (2010 and 2011). I started that job (paid this time!) back in mid-August. Working there, being paid to do what I do, and having so many more responsibilities than I ever did before, I feel just a little bit more like a “scientist.” If that makes sense. I’ll see if I can explain.

When I was a summer lab assistant at UIC, I worked with a wonderful post-doc named Olga who has become something of a mentor to me. She still works in the same department, and her lab room is actually next to mine now. When I have a question about something, I usually go ask Olga. Not only will she help me (or help me figure out where to find the answer to my question, if she doesn’t know), but she does so happily and willingly. Not everyone is like that. Olga taught me to pipette, to set up PCR, to run gels, to culture cells. It was while I worked with her that I discovered my love of bench science and research.

With the wonderful background Olga provided me, as well as what I learned in my post-bac coursework and research at Dominican, I felt I was ready for a position with more responsibility and autonomy. Well, I got it! While there was a bit of a rough start, things are going quite well now. And in that rough start, I learned a great deal about the scientific process, and my aptitude for it.

I was hired at UIC to genotype mice. My department is trying to breed double knockouts (DKOs, for short) of several genes in order to study these genes’ combinatorial roles in lung diseases. Genotyping mice, at least the way we are doing it, involves (in part) two techniques that are quite familiar to me: PCR and gel electrophoresis. I did both of these with Olga, as well as in my Research Methods course at Dominican. But genotyping involves many more steps as well, steps which were new to me. In addition, while I had done both PCR and gels, I had never done them completely from start to finish. By that, I mean from ordering primers to taking the gel photo in the dark room. I had done the middle part – the actual PCR and the gel – but not the initial and final steps. So I definitely had a lot to learn when I started this new position.

So here is the basic outline of the genotyping process:

1. Cut small pieces (about 0.5 cm) of mice tails
2. Digest mice tails to extract DNA
3. Run two rounds of competitive PCR (one for each gene) to amplify the DNA
4. Run a gel to determine whether each mouse is a wild type, knockout, or heterozygote for each gene
5. Analyze results, and hopefully set up new breeding pairs if you get any DKOs

Steps 1 and 2 were brand new to me. But I am proud to say that I am now a mouse anesthesiologist and surgeon! On one of my first days at the lab, my supervisor took me down to the animal facility and taught me how to anesthetize the mice, tag their ears, and snip of a bit of tail into an eppendorf tube (being careful to wipe with ETOH in between each mouse to avoid DNA contamination).

One of my gels, from last week. Pardon  the over-exposure and the line of dNTPs  at the bottom. I'm still working out the kinks with my primer concentrations and still getting the hang of working the darkroom camera ... it's a work in progress, but I'm getting there!

One of my gels, from last week. Pardon the over-exposure and the line of dNTPs at the bottom. I’m still working out the kinks with my primer concentrations and still getting the hang of working the darkroom camera … it’s a work in progress, but I’m getting there!

The tail digestion step was a bit of a debacle to begin with. I started out with a complex and time-intensive protocol that was handed down to me by another post-doc at the lab. It involved using proteinase K and several other reagents, heating the tail tubes in a 55°C water bath for four hours, and then inactivating the proteinase at 95°C for 10 minutes. When you counted prep time (thawing reagents, pipetting, labeling tubes, etc.), the whole process lasted somewhere between five and six hours. And I was getting very inconsistent results. But honestly, I wasn’t sure whether it was the tail digestion or the PCR that was the problem. For example, my primers could have been bad, or I could have been over- (or under-) digesting the tails. I literally worked with this protocol for a month, to no avail. Then my supervisor suggested I go talk to the woman, Debbie, who runs the Molecular Core, where the PCR machines are. I asked her how long she incubated her tails for.

“Oh, I use a kit,” she said, nonchalantly. “Why, what are you doing?” After I gave a brief outline of my method, her co-worker literally busted out laughing and said, “Wow, you’re really doing it old school!”

This “kit” is a miracle: while my protocol was taking five to six hours, the kit takes 30 to 45 minutes. And, as if that weren’t enough, the kit comes with a pre-made PCR master mix that contains a Taq JumpStart antibody which prevents the Taq from activating at room temperature … meaning you can set up PCR on the lab bench rather than on ice! It’s absolutely amazing.

Debbie let me borrow her kit to try it out, and it worked beautifully. My results are now consistent, and I even determined last week that we indeed did have several DKO mice – three males and one female.

While it’s obviously exciting to get results, what’s also exciting to me is the process of science. I faced frustration, and I didn’t give up – I worked on figuring out what was going wrong. Also, one of the things that always amazed me about Olga (when I worked with her those first two summers) was that she always had several experiments running simultaneously, and somehow the timing all worked out. I am learning how to do that as well – how to time my agarose melting, reagent thawing, and PCR and gel running (along with meticulous notebook note-taking) so that I get the most out of my time there.

I work at the UIC lab three days a week. This past Thursday I was working from home on freelance writing. When I woke up that morning, I felt a little sad, and thought to myself, “I wish I were going to the lab today.”

I think that’s a good sign.