How Many Times can a Clone Be Cloned?

“How many times can you clone a clone?” she asked, “Is that even possible?” The rest of the class was filing out of the classroom, but one student stood in front of me trying to get my attention. This was a BIO 101 class and we had finished a discussion on biotechnology two minutes prior.

I stopped coiling my laptop’s charge cable for a second. Sensing an opening, she continued, “So can you make an army of clones or does something happen that prevents this?” I could tell that she thought this was a stupid question. Many great questions feel like this to the asker. They might seem like they have obvious answers, and for all I know, she was second-guessing even having asked it. This was likely worsened by the fact that I found myself unable to respond right away.

As I closed my laptop’s lid, I thought about the deceptive simplicity of her query. How many successive clones can be made from one organism? I told her I would think about it and get back to her. The concept of infinite clones seems like it might be logical. Possibly even useful to some industries. To be honest, aside from thinking about the army of Jango Fett clones in Star wars or maybe Gru’s Minions (they seem asexual – likely clones), it never occurred to me to explore what the real physiological limitations on cloning might be.

So what would successive cloning be like for the DNA of the clones? Are we making perfect digital copies each time, like the way I copy files on a hard drive? Or is it more like when I used to record TV shows on VHS tapes or mixtapes on cassettes when I was a kid – each successive copy drowning in more and more static?

How are clones created?

Clones are created when a parent produces offspring genetically identical to itself. In 1996, researchers in Scotland successfully produced the first animal clone. This clone was Dolly and she was a sheep produced by a process called somatic cell nuclear transfer.

Somatic Cell Nuclear Transfer (SCNT) is a process by which a somatic cell (body cell) is harvested from the animal to be cloned. The nucleus (where all the DNA is stored) is carefully extracted from this cell and injected into the denucleated ovum (female egg cell that lacks a nucleus) of another animal. If the process goes well, the ovum will begin dividing as if it were a zygote. An electric shock jump-starts the division artificially. This zygote then develops into an embryo that can be implanted into a surrogate mother.

Dolly, and every animal clone since, has been created by this process. It is a process that has undergone much refinement since the 1990s, but it is still not perfect. This study found that the success rate of cloning is around 10%. Some common causes of failure are the death of the embryo, a failure of the embryo to implant into the surrogate mother’s uterus, and failure to correctly develop the nutritive placenta for the fetus. This high failure rate is a direct result of the fact that while it is possible to transfer donor DNA into a recipient sex cell, it is not easy to get the DNA to behave in the way a real zygote does. In a zygote formed by sexual reproduction, DNA is an open book. But when an organism develops, cells specialize. These cells can become neurons, skin cells, liver cells, or any other cell. As a cell specializes in one job, it coils the DNA is doesn’t need around proteins called histones. Think about a long thread coiled around tiny spools. The cell leaves the thread it needs to use uncoiled, and coils the DNA it doesn’t need.

Making a clone from the DNA of a mature animal is complicated because even the most unspecialized cells (stem cells) to be found in the body will have some of the DNA locked away like this. The key to successful cloning is finding a way to uncoil the DNA.

Can I create a clone army?

No. Well, you shouldn’t. At this point, science doesn’t have a way to make this happen This doesn’t mean that scientists are not working on it, though. They have a name for what we are talking about. That name is serial cloning. The record for most clones produced from a single animal is over 500. This was published in 2013 in the Journal Cell Stem Cell. The authors of this paper were able to clone a single mouse to 25 generations! This resulted in over 500 mice genetically identical to the original.

cloned mice
Cloned Mice. Credit: Riken

So how did they do this? Typically, any somatic cell used as a nucleus donor is going to have DNA coiled around histone proteins as a method of controlling which genes can be expressed. While the order of bases in DNA is referred to as the genome, the configuration of regulatory factors such as histones is referred to as the epigenome. So, in a cell, the genome is like a recipe book and the epigenome is how you decide what to cook.

The epigenetic configuration of the donor cell’s genes has been a chronic problem for cloning technology, as clones tend to carry on accumulated epigenetic abnormalities from their parents. This team cloned a mouse to 25 generations by exposing the donor DNA in each generation to a chemical called Trichostatin A (TSA). If TSA is added to DNA, it removes certain chemical components from the histone proteins the DNA is coiled around, allowing the previously-coiled genes to be expressed. In other words, it can reset the mouse epigenome, preventing the accumulation of epigenetic abnormalities that would otherwise accumulate.

Before you go off to create an army of cloned bounty hunters, it is important to note that this technique has only worked in mice. Attempts with other animals have struggled clone more than three successive generations. This is because reprogramming the epigenome is not always as simple as breaking histone proteins. There are other methods of genetic regulation (see DNA methylation and gene imprinting) and these regulation methods tend to accumulate as well. This accumulation will result in epigenetic abnormalities which ultimately lead to failure of the clone.

Cloning is Natural

Taking a hike in Fishlake National Forest is a tranquil experience. This national forest in south-central Utah has plenty of attractive features, such as elk, deer, and other wildlife that roam the aspen stands. The aspen trees are the oddity here. As you walk through the forest, you may be unaware that clones surround you.

Aspen Clones. Credit: US Forest Service

The aspens in Fishlake National forest have a name. Some refer to the forest as “Pando”, others the “Trembling Giant”. The reason the name is singular is that this Aspen forest represents the world’s second-largest organism, a quaking aspen that covers 43 hectares and is composed of 43,000 genetically identical trees all connected to a root system underground.

Aspens proliferate by cloning. An underground root system spreads through the soil and produces forests of clones. Some estimates suggest this quaking aspen has been surviving by cloning for roughly 80,000 years.

Cloning is common in plants. Many of the fruit you eat, such as bananas, oranges, and apples, are all clones. They are produced by cutting branches from a plant (a banana tree, for example), and planting it in such a way that a new tree can grow from the cutting. Those eyes that form on old potatoes? Clones that can be planted.

Cloning is not limited to plants. Many species can undergo parthenogenesis. This is the ability to produce offspring without a male genetic contribution. This process has been observed in insects, reptiles, amphibians, and even in some birds. It doesn’t stop there. Some fish, all jellyfish, and all sponges can reproduce without sexual partners if need be. This is not a comprehensive list. Producing clones is rather common in nature, as opposed to the rarity we often think it might be.

You may have noticed a group of animals conspicuously absent from the list above – mammals. Mammals are one of the only taxonomic groups on Earth in which parthenogenesis does not happen.

Mammal cloning

Parthenogenesis occurs when an ovum (unfertilized egg cell) begin dividing as if it had been fertilized. A process of cell division, called meiosis, produces egg and sperm cells. The purpose of meiosis is to create sex cells with half the number of chromosomes as normal. This way, two sex cells can combine their genes to create a new genome. If meiosis fails to reduce the number of chromosomes in the ovum (often by failing to split into new cells), the ovum will have a complete set of chromosomes and behave as if it were fertilized. This behavior leads to an embryo genetically identical to the mother.

Parthenogenesis in mammals doesn’t happen because even though a mammalian ovum with a complete genome can be accidentally produced by meiosis, the embryo will fail to develop. This is because of a type of DNA regulation known as gene imprinting. Gene imprinting activates or deactivates certain genes depending on which parent they came from, be that parent a male or a female parent. Mammals require imprinted genes from both parents to survive. Experiments that have induced parthenogenesis in mice have only succeeded by manipulating the DNA to mimic male imprinting.

Cloning efforts have largely focused on perfecting the ability to clone mammals. This is entirely because mammals do not naturally clone themselves. To answer the original question, it depends on what is being cloned. It is possible to clone a mammal a few times, while aspens can clone themselves for thousands of years. It depends on how the organism regulates its genes. Copying the genome is not the difficult part. Finding a way to reprogram the epigenome is the hurdle. At least for mammals. It seems most everything else has beat us to that point.

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