Dr. O'Neil worked with Eric Sacks on a secret project, whose intent to create a serum to protect people against biological weapons, with four baby turtles and a rat as the test subjects. However, O'Neil realized that his partner's purpose was far from as noble as his own, finding out that Eric Sacks worked for the Shredder and would use their hard work to contaminate the city he wanted to protect. Seeing no other choice, Dr. O'Neil set his lab and research on fire while he and his daughter were in it, preventing Sacks from getting control of any more of their work. His daughter escaped the fire with the animal test subjects, and Dr. O'Neill himself he was shot and killed by his former partner Sacks.
Splinter used Dr. O'Neil as a role model for his relationship with his sons.
Eric Sacks says they considered using rabbits instead of turtles for the mutagen experiences, it can be interpreted as a reference to either Usagi Yojimbo or The Tortoise and the Hare tale.