“Please amplify the adenovirus containing Prox1.”
My professor’s instructions were always short, but they left my mind tangled for days. Why don’t professors ever tell you why they want something done?
Looking back, I think I was his ChatGPT before ChatGPT even existed.
Why Prox1? Why adenovirus? And what on earth was he planning to do with it once I amplified it?These weren’t questions I could ask. As a graduate student, my job was to Google, search papers, and figure it out myself.
Adenovirus Gene Delivery: A Graduate Lab Reflection
In the spring of 2015, I found myself tasked with a seemingly straightforward assignment: amplify an adenovirus containing the Prox1 gene. It was part of our lab’s ongoing efforts to study lymphatic differentiation—but at the time, I wasn’t told that.
All I had was a brief instruction from my professor: “Please amplify the adenovirus containing Prox1.”
Why Prox1? Why adenovirus? What was its destination after amplification? These questions circled in my mind. But as a graduate student, my job wasn’t to ask—it was to search, execute, and trust the protocol.
Looking back, that single task introduced me to the profound complexity of adenovirus gene delivery. In that dimly lit incubator room, with only 293A cells and cross-pattern swirling to keep me company, I wasn’t just propagating virus—I was cultivating identity, curiosity, and a quiet resistance to academic opacity.
Prox1: The Fate Decider of Lymphatic Identity
The lymphatic system—so often overshadowed by the blood vascular network—is essential for fluid balance, immune cell trafficking, and fat absorption. Yet for decades, its developmental origins remained obscure. Then came Prox1.
Knockout mouse models revealed that Prox1 is essential for lymphatic vessel formation. Without it, mice failed to develop any lymphatic structures, retaining only blood vasculature. When overexpressed, Prox1 transformed vascular endothelial cells into lymphatic ones, upregulating LYVE-1, Podoplanin, and other markers.
My task—amplifying Prox1 using an adenovirus gene delivery system—suddenly felt less routine and more symbolic. I wasn’t just working with a gene; I was handling a molecular identity switch.
Learning the Language of the Virus
Back then, I didn’t know the rules of the game. I let 293A cells overgrow. I didn’t recognize the cytopathic effect until I saw floating cells and freaked out. I trypsinized, froze, thawed, harvested, and repeated, trusting that something invisible was working.
The beauty—and burden—of adenovirus gene delivery is its transient, high-efficiency expression. Unlike retroviruses, adenoviruses don’t integrate into the host genome. They crash the party, drop their payload, and vanish. Rapid. Clean. Temporary.
That very temporariness fascinated me. Does transient expression mean less truth? Or does it reflect the ephemeral nature of biology itself?
Technique Meets Existentialism
In the lonely, fluorescent corners of the lab, surrounded by biosafety cabinets and half-filled T25 flasks, I began to think: is a gene truly expressed if it’s not integrated? Is a scientist truly learning if they’re just following orders?
Adenovirus was my teacher. Not just in molecular biology, but in philosophical discomfort. It taught me the difference between running a protocol and understanding a purpose.
That spring, the task was simple: generate enough virus for in vivo experiments. But the meaning wasn’t.
Looking Back: What Was Amplified
In hindsight, the entire episode might earn a single sentence in a methods section. Or perhaps be lost in supplementary data. But for me, it was a defining moment.
I learned how to harvest virus, yes. But I also learned how to question, endure, and reflect. I amplified Prox1 with adenovirus. But in doing so, I amplified more than a gene—I amplified myself.
Even today, when I see a paper on adenoviral vectors or Prox1 expression in lymphatic research, I think of those long hours, the trial-and-error, the isolation, and the discovery.
Because in that dark corner of the lab, I wasn’t just conducting an experiment.
I was becoming one.
🧬 The Beginning of Lymphatics — and Prox1
The lymphatic system is composed of blind-ended thin-walled capillaries and thicker collecting vessels that return interstitial fluid to the bloodstream. About a century ago, Sabin proposed that endothelial cells sprout from embryonic veins to form primitive lymph sacs, which later give rise to lymphatic networks.
For decades, this idea lacked concrete molecular evidence. That changed with Prox1 knockout mouse studies. These mice failed to develop lymphatic vasculature entirely, leaving only blood vascular structures. When Prox1 was ectopically expressed in human vascular endothelial cells, lymphatic markers increased and blood vessel markers declined.
Thus, Prox1 became known as the “fate-determining factor”—the switch that turns a vascular endothelial cell into a lymphatic one. My task was to amplify this very Prox1 gene using adenovirus, and even today, checking Prox1 expression is still the most direct way to confirm a lymphatic identity. It’s like a cell’s name tag.
🔬 The Virus on the Bench — and the Human Behind It
I didn’t know back then that 293A cells shouldn’t be over-confluent. Or that cytopathic effect (CPE) meant it was time to harvest. I detached cells with trypsin, swirled the plate in a cross pattern, and froze and thawed until the virus was ready.
But all of this required hands-on precision. It took time. I wasn’t a machine—I was a graduate student. A role that, in hindsight, feels incredibly undefined. Neither novice nor master, both worker and thinker.
The goal was simple: to generate enough Prox1 adenovirus for later in vivo studies—hoping to see, finally, the formation of lymphatic vessels.
But to me at the time, it felt overwhelming. As if I wasn’t the one conducting the experiment—the experiment was conducting me.
🔁 Adenovirus, Retrovirus, and the Nature of Expression
Eventually, I found myself wondering: Why adenovirus? Why not retrovirus?
Adenoviruses replicate independently in the cell, like plasmids, enabling fast and transient expression. Retroviruses, on the other hand, integrate into the host genome and sustain long-term expression.
Is speed closer to truth?
Or does enduring expression feel more “real” because it becomes part of the cell?
I know it sounds like a humanities question, but I think science hasn’t fully solved it either. It’s a bit like asking: What came first, the chicken or the egg?
I still don’t have a perfect answer.
But I do know this: experiments always balance on a tightrope between what’s true, and what merely looks like truth.
🌱 Looking Back on the One Who Did the Work
Now, this whole episode could be reduced to a sentence in a paper, maybe even a footnote. But at the time, this felt like the most important task in the world.
In that dark corner of the incubator room, while culturing viruses with tired hands, maybe—just maybe—I was cultivating a fragment of biology’s larger story. With my own hands.
That spring, I was amplifying Prox1.
But I was also amplifying my experiment, my belief, and myself.
March 16, 2015
— From a graduate student’s lab notebook
