1. Which kind of cell was used to create Dolly at the Roslin Institute?
Dolly was born on July 5, 1996, at the Roslin Institute in Scotland. A team led by Ian Wilmut and Keith Campbell created her. They used a mammary gland cell from a 6-year-old female sheep of the Finn-Dorset breed [1].
There were 277 tries in total. The only lamb born alive was Dolly [1]. The results were published in the science journal Nature on February 27, 1997 [1]. The idea that a new animal could grow from a cell in a body part was tried before Dolly. The next section looks at frog experiments from more than 30 years before Dolly.
2. Do adult cells keep the genes needed to build a whole body?
As cells grow, they become skin, muscle, or other types. The question was: do they throw away the genes they do not need, or keep them? John Gurdon tried an experiment with frogs around 1962. He put the nucleus of an intestinal cell into an egg that had its own nucleus removed. A healthy tadpole grew [3]. This is considered the first successful example showing that the nucleus of a body cell still has all the genes needed to make a body [3]. Gurdon won the Nobel Prize in Physiology or Medicine in 2012, along with Shinya Yamanaka [3].
Wilmut’s 1997 paper reported that lambs were born from three types of cells: adult mammary, fetal, and embryonic cells [2]. The paper stated that getting a lamb from an adult cell confirmed that the genetic material needed for development is not irreversibly changed during cell differentiation [2]. But how do scientists put the nucleus into the egg?
3. What are the four steps of making a clone by moving a nucleus into an egg?
The process for making a somatic cell clone has four steps [4]. First, take a body cell (like skin or muscle) or its nucleus from a donor animal. Second, take an unfertilized egg from another animal and remove its nucleus. Third, put the donor cell or nucleus into the empty egg and fuse them using electricity or other stimulation. Fourth, place the growing embryo into the womb of a female animal to give birth [4]. The born animal has the same nuclear genetic information as the donor [4]. However, this "same" refers only to the nucleus. Other differences are discussed in section 5.
The steps sound simple, but Dolly was 1 out of 277. Why is the success rate so low? This process is called somatic cell nuclear transfer (SCNT). Whether the nucleus of a body cell can follow development in the same way as the nucleus of a fertilized egg decides success or failure.
4. Why do many cloned cows and pigs die before or after birth?
For cloned cows and pigs, many die before or after birth [4]. The Food Safety Commission thinks the cause is that the embryo with the moved nucleus (the reconstructed embryo) cannot fully regain "totipotency," among other factors. Totipotency is the state where a cell can become any type of cell, like a normal fertilized egg [4]. This is a current explanation, not a proven fact [4].
Interestingly, the offspring of cloned cows and pigs (the next generation) are born through normal fertilization. High death rates before or after birth are not seen in them [4]. Cloning is still a difficult technology. But if an animal is born, is it exactly the same as the original? The next section looks at a cat and at sheep.
5. Why was the cloned cat CC not a perfect copy of her genetic mother?
In December 2001, Texas A&M University in the US announced CC (Carbon Copy), the first cloned pet cat [5]. The genetic mother, Rainbow, was a calico (orange, black, and white). But CC was a tabby cat without orange fur [5]. This shows that the same genes do not always make the same look.
There are also invisible differences. Mitochondria are small parts in cells that make energy. They have their own small amount of DNA. In a study of 10 sheep born by nuclear transfer, including Dolly, the mitochondrial DNA came from the egg that had its nucleus removed. No mitochondrial DNA came from the body cell donor [6]. So, the nuclear DNA is from the donor, but the mitochondrial DNA is from the egg. This state is described as a "genetic chimera" in this study [6]. This method is not just for sheep and cows. In 2018, it was tried with monkeys.
6. How were crab-eating macaque monkeys cloned in 2018?
In 2018, a team at the Institute of Neuroscience, Chinese Academy of Sciences, reported success in cloning crab-eating macaques using the same method as Dolly [7]. When they used fetal fibroblast cells, 6 out of 21 surrogate mothers became pregnant, and 2 healthy monkeys were born [7]. However, when they used adult cumulus cells, 22 out of 42 became pregnant, and 2 were born, but they did not live long [7]. So, successful clones from adult cells were short-lived.
Adding treatments with Kdm4d mRNA and trichostatin A greatly improved embryo development and pregnancy rates [7]. So far, we have used cells from living animals. What about cells from animals that have died?
7. How was the famous bull Yasufuku cloned from frozen cells?
Yasufuku was a famous bull called the "father of Hida beef." He died in 1993. His testes (a male reproductive organ) were stored in a freezer at -80°C for more than 10 years without special treatment [8]. The Gifu Prefectural Livestock Research Institute and Kindai University took living cells from this frozen tissue. The key to the study was being able to grow a few remaining living cells in a lab [8].
Using these cells, a cloned cow named Nozomiyasufuku was born. The Secretariat of the Agriculture, Forestry and Fisheries Research Council selected this achievement as one of its Top 10 Research Topics of 2009 [8]. We have looked at sheep, cats, monkeys, and cows. Where can you look for more information?
8. Where can you read more about somatic cell cloning?
In Japanese, you can read the Food Safety Commission’s Q&A on somatic cell cloning [4] and the Agriculture, Forestry and Fisheries Research Council’s top 10 topics [8]. The sources used here do not explain why CC’s coat color differed from Rainbow’s; that question is beyond the scope of this article. For original records, Dolly’s birth is documented by the Roslin Institute [1] and the Nature paper abstract [2]. The Food Safety Commission’s Q&A explains the process and safety [4]. Note that the Q&A explains the situation at the time it was written [4].