Thirty years ago, researchers accomplished something that had previously belonged only to the realm of science fiction: they cloned a complex living creature — Dolly the sheep.
In cloning, a cell is taken from the body of the donor animal — the biological mother or father of the cloned offspring — and its nucleus is removed. The nucleus contains most of the hereditary material, apart from the genetic material found in organelles called mitochondria. This nucleus is then inserted into an egg cell taken from another female, after that egg’s own nucleus has been removed. The egg cell is grown in a laboratory dish during the early stages of embryonic development, and the resulting embryo is eventually implanted into the uterus of a third female, where it develops through a normal pregnancy. The animal born through this process is almost completely identical to the donor animal.
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In the cloning process, the nucleus is removed from a cell taken from one animal and inserted into an egg cell from another female, whose own nucleus has been removed. The resulting embryo is implanted into the uterus of a third female. Illustration of sheep cloning
(Illustration: Shutterstock, grayjay)
Dolly, the first cloned mammal, became a celebrity — which probably also hastened her death. Since then, scientists have successfully cloned many other mammals, including mice, rats, cattle, horses, dogs, cats, and even macaque monkeys. Yet despite these achievements, cloning remains inefficient and prone to problems. To better understand what happens during cloning — and how far the technology might be pushed — researchers set out to ask a deceptively simple question: is there a limit to how many times an animal can be cloned, generation after generation?
Finding the limit
In 2013, developmental biologist Teruhiko Wakayama and his team at the University of Yamanashi in Japan published a study that followed 25 generations of clones descended from the same female mouse. They found that repeated cloning did not reduce the efficiency of the process. In fact, efficiency increased, from a 5–8 percent success rate in the early generations to 10–14 percent in the later ones, even though the researchers used the same technique throughout. They also found no signs of declining health in the cloned female mice across the generations.
This raised the question of whether cloning could continue indefinitely. Recently, in a new study published in Nature Communications, the researchers showed that there is, after all, a limit to how many generations can be cloned. They found that from the 27th generation onward, cloning efficiency began to decline gradually, until the process reached a dead end in the 58th generation. By the 57th generation, efficiency had fallen to just 0.6 percent, and in the following generation, no offspring survived for more than a day after birth.
In total, about 1,200 female mice were born across all the generations, all of them clones of the same female mouse from 2005. They were almost completely identical to the original mouse in both appearance and genetic material. Their average life expectancy remained about two years through generation 56, similar to that of non-cloned mice. The females of generation 57, however, died younger: one at two months old and another at six months old.
Cloned mice show several unusual traits that distinguish them from mice born naturally. For example, their fetuses and placentas are larger, as is the area where the placenta attaches to the uterus. However, the researchers did not find that these abnormalities worsened from generation to generation. They also found no differences in the early embryonic developmental stages, before implantation in the uterus.
A fine thread of genetic errors
Genetic sequencing of the female mice revealed that, over the generations, more and more errors — known as mutations — accumulated in their genes. These errors affect how efficiently eggs develop into embryos. In in vitro fertilization experiments using sperm from ordinary mice, the researchers found that 97 percent of fertilized eggs from normal female mice developed into embryos.
By contrast, only about 40 percent of fertilized eggs from 25th-generation cloned female mice developed successfully into embryos. By the 53rd generation, that rate had fallen to just 20 percent. From this, it is reasonable to conclude that the accumulating mutations interfere with normal embryonic development.
The researchers found that, in each generation, the clones accumulated an average of about 70 point mutations — substitutions of a single building block in the genetic sequence — for a total of about 3,700 mutations by the final generation. This rate of genetic change is three times higher than in ordinary reproduction.
In addition, about 80 large-scale changes accumulated in the genetic material of the cloned mice’s cells. These included 16 cases in which long segments, consisting of tens of thousands of genetic building blocks, were deleted, as well as cases in which long segments moved from one chromosome to another, and even the loss of one of the two X chromosomes — the female sex chromosome. These changes began to appear only from the 25th generation onward. It also became clear that most of the harmful mutations accumulated in the later generations.
The clones’ problems were not limited to the genetic material in the nucleus. The researchers also examined what happens when the nucleus from a normal female mouse’s cell is implanted into an egg from a 56th-generation cloned female mouse. If the only problem had been the condition of the genetic material in the nucleus, then no developmental problem should have appeared in these embryos, compared with the reverse experiment — implanting a nucleus from a cloned female mouse into an egg from a normal female mouse.
However, in both cases, embryo formation was inefficient, reaching only 7–8 percent. The conclusion was that, in addition to mutations in the genetic material of the cell nucleus, changes may also accumulate in mitochondrial DNA, and perhaps also in the composition of the substances and organelles in the egg cell’s cytoplasm.
Interestingly, when female mice from the 55th generation were bred with males, they gave birth to healthy offspring. Moreover, all the unusual traits seen in the clones disappeared in their grandchildren, which were born through natural fertilization. In other words, even cloned animals from almost the very last generation could still produce healthy offspring. The researchers explain that the fathers’ normal genetic material prevented most of the mutations accumulated by the cloned females from being expressed and impairing the offspring’s function.
It is therefore possible to clone many generations of mice from a single mouse, but over time, more and more changes and defects accumulate, eventually making further cloning impossible. The limit reached in this experiment was 58 generations, but that number is arbitrary. It depends on the number and type of mutations that accumulate — specifically, on when a destructive mutation appears. In another experiment, the limit might have been reached earlier or later
Likewise, the rate at which genetic changes accumulate in clones, and the types of mutations involved, may vary from one mammal to another. It is therefore difficult to know how far findings from cloned mice can be applied to dogs, cows, monkeys, or humans.
The researchers identified two main processes that lead to the unusual accumulation of mutations. The first is the accumulation of large-scale mutations across broad regions of the genome — something that can also occur naturally, though rarely, in gametes, the sex cells such as eggs and sperm, which are usually better protected than ordinary somatic cells.
The second is the absence of recombination in the somatic cells used for cloning. During the formation of gametes, recombination allows segments of genetic material to be exchanged between chromosomes, which can help repair some accumulating mutations. Because this process does not occur in cloned somatic cells, more mutations accumulate over the generations. To succeed in cloning animals repeatedly in the future, researchers will need to find ways to address the problem of mutation accumulation.