In February 1997, researchers in Oregon produced two Rhesus monkeys using laboratory techniques that had previously worked with frogs, cattle, and mice. They collected eggs from and sperm from adult monkeys and allowed fertilization to occur in plastic laboratory dishes, a standard in vitro fertilization (IVF) technique. After the fertilized eggs had divided several times to produce very early embryos called blastocysts -- tiny balls of only 6 to 12 cells -- the researchers transferred one of these cells into an "enucleated" oocyte. They allowed the oocyte to divide several times and then placed it into the uterus of a surrogate mother, where embryonic development occurred.
This was the first time that researchers had been able to use a nuclear transfer procedure -- taking the nucleus from an embryonic cell and fusing it with an enucleated oocyte -- to generate monkeys. Everyone paid attention to the research because monkeys are so closely related to humans, at least in evolutionary terms. People reasoned that if scientists could clone monkeys, they might also be able to clone humans.
Don Wolf, of the Oregon Regional Primate Center in Beaverton, headed the research team that generated the monkeys. The most important difference between the monkeys and Dolly is that unspecialized embryonic cells were used to create the monkeys, whereas a specialized adult cell was used to create Dolly.
The laboratory techniques used to generate the Rhesus monkeys are illustrated here.
To generate the monkeys, the Oregon researchers used standard in vitro fertilization techniques. They collected sperm and eggs from adult monkeys and allowed fertilization to take place in plastic laboratory dishes, called an "in vitro" environment. The eggs that became fertilized began to divide until a blastocyst or ball of 6 to 12 cells resulted. The researchers removed the coating (zona pellucida) that surrounds the blastocyst and separated the cells. Each cell carries the same genetic material -- because each developed from the same fertilized oocyte. These embryonic cells became the potential "donor" cells for the cloning procedure.
Meanwhile, the researchers injected female monkeys with hormones to make them ovulate. They recovered several mature oocytes from the monkeys and removed the nucleus from each egg cell using a tiny glass needle. The process is called enucleation. The enucleated oocytes served as the recipient or "host" cells for the cloning procedure.
To create the monkey clones, the Oregon researchers used a mild electric current that caused a single donor nucleus (from an embryonic cell) to fuse with the host oocyte (an enucleated egg). This process is called nuclear transfer, and it generates an egg cell that behaves as if it has been fertilized by a sperm cell. The artificially "fertilized" eggs divided by mitosis and became blastocysts in plastic culture dishes that contain a nutrient-rich liquid. The dishes and blastocysts were kept warm in an incubator.
In the final step, the researchers transferred two or three blastocysts into the uterus of a surrogate mother monkey. If the procedure works as planned, the surrogate mother carries at least one of the dividing embryos through a normal pregnancy and gives birth to a healthy infant Rhesus monkey.
Most embryos produced by nuclear transfer do not survive these procedures. In the Oregon experiment, only two monkeys survived. They were generated from different donor blastocysts and so are not clones of each other. Instead each monkey is a clone of the original blastocyst that had developed from a fertilized egg.
