Chapter 2

December 2006

The following week, when Ross rolled in to the lecture hall with Hanna once again in tow, the room was packed. Word had traveled fast about Ross's intriguing combination of scientific inquiry into the genetic underpinnings of associative learning and a wildlife-park circus act. He had given a number of research assistants something that never failed to attract the attention of budding scientists eager to roll up their sleeves: A chance to stop reading about somebody else's work and start doing some of their own.

He opened the class with another amazing performance by Hanna, this time demonstrating her mastery of the abstract concepts of "similarity" and "dissimilarity," both as they related to colors and specific items. Ross presented the bird with different items, such as a plastic chip and a small toy truck of a similar color, and asked her to identify if they were the same or different. After correctly responding "different," she was then asked if they were the same or different color, and she was able to correctly respond "same." He repeated the demonstration using various items and colors, with Hanna racking up a respectable accuracy record--just over 80 percent across-the-board.

Once show-time was over, Ross turned his attention to a half-hour discussion of the reproductive biology of the African Grey parrot. One of his handouts included a detailed anatomical drawing of an avian oviduct, showing how the mature ovum (the familiar yoke of a bird's egg) is released into the infundibulum (where fertilization takes place), and then progresses down the oviduct where progressive layers of albumen (egg "white"), membranes, and finally, the hard, calcium-rich outer shell are added. When he was done he apologized for the high-school biology lesson and promised its relevance would become clear shortly.

After a break, Ross switched gears and started presenting concepts concerning the use of vectors in effecting germ-line gene transfers.

"What's a vector?" he asked. He nodded in the direction of a student who looked like she knew the answer.

"It's a virus--generally a genetically engineered virus--used as a delivery vehicle for a particular gene into the genetic structure of the target organism. It's sort of like a very specific, targeted infection."

"Very good. Why do we use viruses? Someone else ... yes, over there."

"Viruses already have the ability to deliver their genes to cells, generally in a pathogenic manner. We just take advantage of that ability, and re-engineer the virus to remove the disease-causing genes and insert the gene we need to be transferred." "Excellent! Somebody's been doing the reading. Any major problems with vector transfers?" He pointed across the room.

"Vector viruses can cause toxicity, immune and inflammatory responses, and possible gene control and targeting issues," the student responded, referring to his notes.

"Ah! Targeting issues? What's a targeting issue?"

"Position effects!" three students said simultaneously

"Very good. Any other available methods of gene transfer?"

"Pro-nuclear micro-injection," answered the guy with the motorcycle helmet, sitting in the front row this time.

"That's right. We use a fine glass needle to inject the DNA sequence we want to transfer into the nucleus of an already fertilized mammalian egg cell. Problems?"

Blank stares.

"The technique has never been tried on birds."

Professor Ross nervously glanced over the shoulder of the surly microbiology lab technician. Hunched over a cluttered stainless steel table, the technician was peering through a powerful microscope set over a Petri dish. Using a slender glass micro-pipette, he plucked a cluster of African Grey sperm cells from their bath of Luria Broth. The clear, yellowish-beige soup was infused with the Sindbis virus--the viral vector selected for the task--"enhanced" with a copy of the NR2B gene.

"Why can't you work on Doogie mice like everybody else?" The technician glanced at the clock on the wall. He had one more procedure to complete before he could call it a day.

"Been there, done that. Mice don't talk." Ross's doctoral dissertation had been built around an experimental protocol involving Doogie mice

"Do you have ANY idea what a royal pain-in-the-ass this is?"

"Humor me."

The technician sat back on his stool and looked at Ross. "Okay, these suckers should be ready to go." The genetically enhanced sperm cells would be used to fertilize an ovum surgically removed from a parrot hen.

"How about the egg we fertilized yesterday?"

"Good to go," the tech responded.

The facilities at Rockefeller University were among the, if not the, best in the academic world, with more than 70 state-of-the-art laboratories catering to all aspects of the life sciences. Overlooking the occasionally scenic East River, the beautifully landscaped, 15-acre campus was located at York Avenue and 65th Street on Manhattan's Upper East Side. Ross had a one-bedroom apartment in the adjacent Scholar's Residence, on East 63rd, connected to the main campus by a convenient footbridge. He rarely ventured far from the protective academic enclave, except to pick up some groceries at Gristedes on 1st Avenue, or visit the hot dog vendor by the M31 bus stop by 64th Street.

"Are you guys ready?" Dr. Lea Perrera asked, sticking her head out from an adjoining room. Perrera, dressed in green surgical scrubs, was an attractive 40-something veterinary surgeon specializing in avian treatment, a relatively rare specialty in the New York region. As such, she was in big demand, and did all the work at the Bronx Zoo and the other area zoological parks, as well as getting a ton of referral work from other, less specialized, veterinarians.

"Is the patient ready?" Ross asked.

"Prepped and anesthetized," she responded. "She's not intubated, so we really shouldn't try to keep her under for more than 15 minutes or so. Let me know when you're ready and I'll open the incisions."

The technician snapped on a fresh pair of latex surgical gloves, then selected a larger, sterile pipette and retrieved a Petrie dish containing the parrot egg, a tiny, yellow egg yoke less than a quarter inch across. It had been fertilized, in vitro, with NR2B-enhanced sperm the day before. He carefully plucked the egg from the dish with the pipette.

"Cut," he said, unceremoniously.

Perrera disappeared into the operating room, followed by Ross and the technician. The patient, one of six African Grey hens that would be implanted with enhanced eggs, was laying on her right side. She was covered with a clear surgical drape, with a small section of skin, plucked clean of feathers, exposed. A small, malleable rubber face mask was secured over her beak.

"How do you like the facilities?" Ross asked the surgeon.

"Top shelf," she responded. "The best small-animal facilities I've ever seen. Even the Bronx Zoo doesn't have a thermal support table like this. And not many places have a sevoflurane vaporizer ... it offers all the advantages of isoflurane with even more rapid post-surgical recovery. That's very important in our case here, where we want her up and laying eggs tomorrow." She slipped on a mask and a pair of latex gloves and adjusted the surgical drape on the tiny patient. "Are we ready to go?" She flipped on the switch to the endoscope monitor.

Ross and the technician nodded.

She selected a small brush, dabbed it in a vial of clear fluid, and picked up a pair of tweezers in her other hand. She carefully brushed the substance on two tiny scars on the bird's side.

"What's that?" Ross asked.

"A solvent," she answered. "I'm going in through the same incisions that I opened yesterday to remove the egg."

"Poor little birdie," Ross said.

"Oh, she'll be just fine..." She gently pried open the incisions with the tweezers and carefully plucked away the few flaky remnants of the tissue glue still surrounding the openings. "Okay," she said, mostly to herself.

Reaching for the endoscopic equipment, she slowly inserted a probe into the lower opening. She maneuvered it gently, looking up at the monitor to ascertain her position inside the unconscious parrot's abdomen. "There's the infundibulum ... Okay, hand me that pipette, please." She held out her left hand. The technician handed her the sterile tube containing the fertilized egg.

She inserted the pipette into the upper incision, again watching the monitor to track her progress. When she was satisfied with her position, she squeezed the rubber bulb just enough to release the egg into the opening to the bird's oviduct, and stood quietly for a moment, studying the monitor to be sure everything looked as it should. Satisfied, she removed the pipette and, after studying the monitor for another minute, removed the endoscope.

"That's that!"

"That's all there is to it?" Ross asked.

"That's all there is to it," Perrera dabbed the incisions with a cotton ball and prepared to re-glue the tiny holes in the bird's side.

"Now you're going to have to keep an eye on her, on all of these birds," she told Ross. "If it looks like they're going to start picking at the incision, we'll have to put a collar on them until it heals."

Ross nodded.

Dr. Perrera finished securing the incisions with the surgical super-glue. She let it set for a moment, then turned to the anesthesia vaporizer and switched off the sevoflurane, allowing the bird to breath pure oxygen for a moment before removing the face mask. She removed the drape and, gently lifting the unconscious parrot, placed her in a recovery chamber, which was oxygen enriched, and heat and humidity controlled.

"Make sure her cage is ready ... she's going to be hungry when she wakes up."

Ross studied the sad-looking bird, lying helpless at the bottom of the portable recovery room. "Is she going to be in any shape to lay that egg tomorrow?"

"She should be perching within the hour. That egg will take about 24 hours to work its way down the oviduct. Make sure the bottom of her cage is clean and check for that yoke ... in the worst-case scenario she may just excrete the yoke with no shell, but I doubt it. She's going to be a little shaken up--this is her second surgery in as many days--and she's certainly not going to sit on that egg ... you know that, right?

Ross nodded. "Greys won't normally sit on just one egg, in any case. It'll be removed to an incubator."

"Right. She should be okay. Just keep a close eye on her and make sure she doesn't pick at those incisions." The vet cleaned up the surgical work area and laid out fresh instruments.

"Are we ready for the next patient?"

The bird recovered fully, and, right on schedule, laid her egg. The remainder of the week was a blur of genetically enhanced viral soups, additional surgical and in-vitro fertilization procedures, recovering birds, and anxiously anticipated and gleefully welcomed parrot eggs. Six birds received the procedure; six eggs were laid.

An additional six eggs were taken from birds that had not received any genetic enhancement, to be used as a control group for future experimental protocols. All of the eggs were marked accordingly, and placed in the incubator for their approximately 30-day journey from embryo to baby bird.

"Congratulations, Professor Ross. I understand that all of your surgical procedures worked as hoped. Six eggs? Is that correct?" A mild-mannered, brilliant and highly disciplined man, Dr. Pradesh was wearing his ubiquitous white lab coat. He waved Ross to a chair across from his desk.

"Yes, Professor," Ross responded, returning the formality. He still hadn't gotten used to Dr. Pradesh, his Ph.D. mentor, addressing him as "professor." "They still have to be DNA-tested to be sure that the gene transfer was successful, but yes, the in-vitro process seems to be successful so far."

"Very good. And how is your guest lecture series going?"

"I'm doing okay. The students seem attentive and generally interested. And Hanna is doing great."

"Ah," he smiled broadly. "Everybody likes the talking bird."

Dr. Sanjay Pradesh, Professor of Neuroscience and head of the Laboratory of Animal Behavior, knew a little something about talking birds, or, more specifically, the neuroanatomy of brain circuits used for vocal learning and song production in birds. Under his direction, his lab had done pioneering work, attempting to unravel the biological structure underlying the complex repertoire of sounds that birds use to manage their daily lives. His laboratory bred most of its research animals (canaries, zebra finches, and now, for Dr. Ross, African Greys), and operated a Field Research Center in Millbrook, N.Y., in rural Dutchess County--1,200 acres of natural habitat 80 miles north of the NYC campus.

A slight, dark-skinned man, Dr. Pradesh was largely bald and wore delicate gold-rimmed glasses. He clasped his hands together--he had willowy, feminine, surgeon's hands--and placed them on his lap.

"So Dr. Ross," he began, addressing the business at hand, "have you finalized your budget requests for the second phase of the study?"

"Yes sir," Ross handed three sheets of paper across the desk. "It's essentially the same as we've previously discussed. I've firmed up the timeline issues and adjusted the budget requirements accordingly."

"Yes, very good." Pradesh glanced at the number at the bottom of the third page. "Still very ambitious, I see."

Ross was one of four Postdoctoral Fellows associated with Pradesh's lab. As senior researchers, they were normally assigned a postdoctoral associate along with two research assistants. Ross was requesting six.

Ross knew what Pradesh was looking at. "I'm going to need those extra bodies. It's the only way I can get 12 birds trained as quickly as we need to. We've got to cover vocabulary, then word/object associations, and then word/abstract concept associations. And that's just to get all of the birds to baseline before we start the experimental protocol..."

"I understand. But must you use the Putnam County facility? The commuter train fares are so very expensive. And we've already got our university facility in Millbrook, with Science Department members going back and forth. Now you want us in Putnam, too."

Truthfully, Ross didn't have to use the Hudson Valley facility; he wanted to use it. "I know. I know it's expensive. But it will be much better for the birds. It's much quieter, much less distracting for them. It'll be a lot easier for them to bond with their trainers and to learn to talk. A couple of the assistants have cars and we're hoping to save a few bucks that way. But I thought I should budget for the worst-case scenario."

"Ah ... that you've done very well." He gently placed the papers down on the desk. "Very well. I don't need to remind you that all of this has given this study, and you, an extraordinarily high profile around here. You understand that, I assume?"

"Sure do, Professor."

Dr. Pradesh was a remarkably intelligent man with a very modest ego. Having spent his entire academic and professional career surrounded by colleagues of equally remarkable intelligence, he never felt the need to either flaunt his own insight or overstate--or even state, for that matter--the obvious. In this case, the obvious meant that equally brilliant researchers would be denied budget dollars for equally compelling research projects as the result of Ross' ambitious wish-list of university resources.

Ross knew what was expected of him.

Founded in 1901, Rockefeller University had been home to 23 Nobel Prize winners, 19 Lasker Awards recipients, 12 National Medal of Science winners, 32 members of The National Academy of Sciences, 14 members of the Institute of Medicine, and 13 Gairdner Foundation International Awards recipients. This was, he knew, well beyond "publish or perish." At Rockefeller University, it was publish, bring fame, world-class accolades and fresh grant money to the organization and to your laboratory head, or perish. Or at least have your budget sliced to ribbons.

A few steps beyond Dr. Pradesh's office, Ross paused for a moment. He had forgotten to mention the voice mail he'd received that afternoon. Some reporter, Jerry Richter from the New York Post, of all places, had left a cryptic message about comments on ongoing research at the university. The only reason he knew Richter's name was from a stupid article on stem cell research, with that goofy-looking little picture of the author's round face. Someone had brought it in and passed it around for laughs. It was so wrong in so many ways no one was sure if it was meant to be serious or just a parody piece. Ross had no intention of calling him back, but thought he should bring it to Dr. Pradesh's attention, just to be on the safe side. Public relations and all that.

It'll wait ... Ross decided.