In this article we will cover:
- What are the main health issues with cloned dogs?
- What is reprogramming and what does it have to do with cloning
- What is Imprinting and how could it affect cloned animals?
- More possible problems: Mitochondrial heteroplasmy
- The telomere concern in cloning
- What about the mutations?
We recommend you first read our article: <how to clone a dog. Recipe included>
What are the main health issues with cloned dogs?
Dogs have already been cloned in this decade, especially working dogs. We know much more about health issues of cloned animals from farm animals, particularly in cattle and sheep compared to dogs because farm animals have been cloned by the thousands already. This is because farm animals are cloned with commercial production objectives in mind.
Most of the health problems that occur in cloned animals have to do with developmental failures. The most common problem is embryo resorption and fetal losses. In other artificial reproduction processes such as in vitro fertilization, embryo resorption or fetal death occurs during the first third of pregnancy. it is interesting that in cloned animals, fetal death occurs throughout pregnancy, but mainly towards the last third or early in neonatal life.
The early loss of these cloned animals is mainly due to developmental abnormalities.
Among this we can summarize:
- High weights at birth (e.g., in the case of Dolly the sheep)
- Lung problems
- Cardiovascular problems
- Immune system defects
- Kidney problems
- Liver problems
- Joints Malformations (like <arthrosis>, among others); (again, like in Dolly the sheep)
- Problems with the placenta (often), as a cause of abortion during pregnancy
In short, many errors can occur during the embryological period leading to a faulty formation of organs (more about this below).
An artificial reproduction technique in animals a little more natural than cloning is in vitro fertilization, which consists of placing sperm cells with an egg in a culture dish so that sperm can fertilize it. The fluids in which these cells are suspended (culture media) are artificial and the embryos obtained from this in vitro fertilization technique also suffer from some of the problems that occur with cloned animals, mainly high weight at birth.
High birth weights can result in problems with delivery (called “dystocia”) which can be dangerous for both the mother and the fetus. Cesarean section surgery is usually necessary. This has led to the belief that the culture media used to create embryos artificially or even clones may not be the most suitable for keeping the cells involved in these artificial reproduction processes and they are constantly being investigated by scientists for optimization. This has been widely observed in cows. However, cloned dogs are born only slightly heavier than naturally bred dogs, so problems at birth are not frequent and they have normal growth patterns.
Early embryos are extremely susceptible especially near the time of implantation in the mother’s womb. Since these embryos are susceptible at this stage, any disturbance (such as defects brought about by the cloning technique) can have serious consequences for future embryological development.
So, in a closer look, what are the main issues that cause problems with cloned animals?
The health problems that occur in cloned animals are caused mainly by two factors:
Reprogramming and cloning
Let’s take a look at reprogramming. The DNA that comes within the nucleus transferred during the <cloning procedure> is adapted to the function of the cell that it originated from and must adapt to its new function: to direct the fate of now an embryonic cell. For example, if the cell of the dog that we wish to clone comes from his skin, the nucleus of this cell was in charge of orchestrating the functions of a typical skin cell, that is to say, the production of proteins like keratin located inside these cells and in the superior layers of the skin.
Now, once this nucleus has been transferred to a previously enucleated egg from a female dog, an embryo is generated which at this moment is composed of only one cell and which from now on must have the functions of growth of an embryo and generation of the future organs. It is then necessary that the nucleus, which was once a skin cell and is now the nucleus of an embryonic cell, to be reprogrammed. If you want more details on how this is done visit our article <How to clone a dog. Recipe included>
In the example we had mentioned, the transferred nucleus must be reprogrammed so that the genes coding for skin proteins are switched off while the genes coding for proteins in an embryo must be switched on.
This becomes even more complex if we take into account that the cells of an early embryo are pluripotent. That is, they have the capacity to generate all the tissues and organs of the adult dog.
In the case of our dog, we would like to clone, this reprogramming must be artificially done in the laboratory. As this is an unnatural process, deactivation of skin genes could be incomplete and the products of these genes (proteins) might interfere with the normal development of the embryonic cells.
This is just one example of how early embryo development could fail and result in the death of the embryo long before the date of birth. However, there are many other ways in which embryonic development can fail in a cloned animal because of problems in reprogramming. The cloning process in these early stages can go awry in many ways.
In natural dog reproduction and in general of mammals, this process of reprogramming occurs spontaneously, even long before the formation of the embryo. It is not necessary for reprogramming to occur in the embryo resulting from natural reproduction, because the gametes (sperm and eggs) have already been reprogrammed during their formation in the past. That is, in other words, both the sperm and the egg have already had their genes irrelevant to embryonic development inactivated and the genes needed for the embryo to grow and develop normal organs activated.
Imprinting and cloning
Imprint is a somewhat mysterious phenomenon of which there are still many details to be known. At any given time in any cell in a dog’s body not all the genes in the total genome (approximately 19.000 genes) are activated. Only the genes necessary to perform the specialized activity of each cell will be activated. Genes that do not need to be functioning at any given time are turned off. This gene inactivation occurs through DNA (genes) chemical binding to certain biochemical markers.
So the genes in a muscle cell will be oriented to the production of muscle proteins, the genes in a neuron will produce neuron proteins, and so on. This specific genetic pattern of switched on genes vs switched off genes, specific for each specialized cell type is known as imprinting and this is a field of biology studied under the name of epigenetics.
Every new individual, e.g., a puppy, received a copy of every gene from the father (through sperm) and another copy from the mother (through the egg). However, these copies behave differently depending on whether they come from the mother or the father. The mother’s imprinting is different from that of the father’s so the copies of genes coming from the mother will not necessarily be on or off in the same way as in the father. The combination of the mother’s and father’s imprint pattern generates the embryo’s imprinting as a whole. When the imprinting “information” of a newly cloned embryo comes from a somatic cell (like a skin, mammary gland, or other cell sources) and not from the union of gametes (sperm and egg), the result may be an embryo with abnormalities that may eventually die. Inadequate imprinting is also responsible for the general errors in the formation of the placenta that can also lead to fetal death, as well as abnormal fetal sizes.
This occurs in cloned animals because the <cloning technique> involves the transfer of a nucleus from a somatic cell (for example, from the skin of the mammary gland of the ovary, among others) and this somatic cell has normal imprinting that has been modified through the life years of the animal that will be cloned as this cell has been dividing many times already. Each time a somatic cell divides (e.g., during normal organ growth), errors can occur in the regions of the DNA that have imprints. The more divisions occurring, the greater the likelihood of these errors to happen. So, when the nuclei of the somatic cells are transferred during the <cloning procedure> these abnormal imprints are also being transferred (this also produces errors and gradual organ malfunction during <aging> of dogs and other mammals, including us).
To make things worse, the culture media used to maintain the cells involved in <the cloning procedure> can affect imprinting (remember, the molecular marks that switch off some genes and leave others unmarked and switch on), aggravating the problem and the possibility of generating abnormal embryos that may die during development.
Talking about dogs, the inadequate imprinting could then be responsible for the <slightly larger size of cloned dogs> compared to those breeding naturally.
Other imprinting problems can arise from mechanical or chemical manipulation of the cells involved in <the cloning procedure>.
It has also been pointed out that in addition to the problems of embryonic death, defective development of the brain and mental function can occur in cloned animals. Fortunately, this has not yet been found in cloned dogs.
More possible problems: Mitochondrial heteroplasmy
Mitochondria are organelles present in all mammalian cells and are responsible for such an important function as it is energy production. Interestingly, mitochondria have their own DNA (genes) for the production of their own proteins and it is believed that millennia ago they were bacteria which were swallowed by primitive cells of that time and which came to have a relationship of mutual interest.
Another curious fact is that all mitochondria are exclusively inherited from the mother (the mitochondria present in the sperm are destroyed when the sperm penetrates the egg during the process of fertilization). In this way, mitochondrial genes are inherited only from the mother while nuclear genes are inherited from both the father and the mother. When the cloning technique involves a fusion step between the somatic cell of the animal to be cloned and an egg (it could be done differently, for instance <by injection of the nucleus into the egg>), the mitochondria of these two cells will be mixed (which does not occur in natural reproduction). This is called mitochondrial heteroplasmy and could generate compatibility problems that lead to embryonic cell death.
So far, it is thought that resulting from a <cloning procedure>, the first single cell of the embryo (the zygote) is responsible for getting rid of the few mitochondria that could come from the somatic cell. Another concern is that the proteins that make up the structure of the mitochondria are encoded partly in the mitochondrial DNA but also by the DNA in the nucleus. For this reason, in any normal cell, there must be coordination between the mitochondria and the nucleus for the synthesis of proteins for the mitochondria. When this coordination does not occur, there is a fall in energy production and the cell may die. This disarray that might come from a somatic cell nucleus being transferred to a cell with different mitochondria during <the cloning procedure> was initially a concern for scientists, but so far there is no evidence of this decoupling in dogs or cloned animals in general.
The telomere concern in cloning
The telomere is a portion of each of the DNA containing chromosomes. Telomeres are located at the cap of each chromosome. Each time a cell divides the telomere is shortened and this has been associated with the aging of cells and therefore the aging of animals (if you were a scientist and would like to know how aged a cell is, a good idea would be to measure the length of the telomeres). For example, a <senior dog> will have shorter telomeres and consequently, shorter chromosomes than younger animals.
In older dogs and other animals, too, when the telomeres are shortened as the cells divide, the likelihood of damage to genes near these telomeres increases. This can lead to cell death and problems in cell division such as cancer, common at an <older age in dogs>, and other animals (including us).
When research about animal cloning began, scientists were concerned about using somatic cells that might have their telomeres already shortened (normal tear and wear, we would say). It was thought that, if for instance, you wanted to clone a 6-year-old dog and you used a cell from its skin, the length of its telomere obviously corresponded to that of a 6-year-old dog and it was then thought that a clone of this dog would be born with a metabolic “age” equivalent to that of a 6-year-old dog.
Although this was not thought to be so literal at first, it was believed that dogs and other cloned animals could suffer from premature aging due to this telomere issue because of the transfer of cells with “aging” telomeres. In other words, the somatic cells would be prematurely “old” or in scientific terms, senescent.
The experience with cloned animals, which comes mainly from working with livestock, has made it clear to us that this telomere problem is not as serious as we thought. This problem can be overcome by wisely choosing the type and origin of the somatic cell to be used in the <cloning process>.
In cattle, it has been observed that once the somatic cells of the animal to be cloned combines with an enucleated egg, the resulting embryo produces high quantities of an enzyme called telomerase that is responsible for reconstructing (enlarging) the telomere which would restore the telomere to its normal condition after cloning, especially during embryological development. So, it seems like most cloned animals get their telomeres recovered during embryo development and this should not be a big concern when we think of cloning a dog or any other animal.
What about the mutations?
Could mutations be the cause of the health problems that occur in cloned animals?
Mutations are defects in the normal DNA sequences of cells (let’s remember that DNA is made up of nucleotides with normal and specific sequences). Mutations are more likely to occur in cells that divide frequently in the adult animal, such as skin cells, gut cells, and glands like the prostate.
Cells that accumulate mutations can die or become malignant cells. In cloned animals, there is a danger of accumulation of mutations that could cause the embryo to die. This is because the somatic cells taken from animals to be cloned have already divided a certain number of times. For this reason, the choice of cells that tend to divide very actively to be used in the cloning technique should be avoided. For example, skin cells have been used to generate cattle clones. Skin cells are normally highly exposed to sunlight. Light from the sun contains some high-energy wavelengths such as ultraviolet rays, so energetic that they can pass through the layers of the skin and reach to the very nucleus of the cell, where they cause permanent chemical changes in the cell DNA, i.e. mutations. If any of these cells are used <in the cloning procedure> (with accumulated mutations), defective cloned embryos could be generated that could eventually die.
Here again, we will mention the use of culture media in the cloning technique, which, not being 100% optimized, could generate mutations in the DNA of the cells involved in the creation of the clone. Many of these theoretical possibilities that generate concerns are sometimes not fulfilled in real life. There is an interesting case of several healthy cloned calves that were cloned from skin cells taken from a 17 years old bull! Amazingly, these skin cells (remember, with high previous exposure to sun UV rays) were subjected to culture media for a long period (3 months). Under these kind-of-adverse conditions, normal clones were produced. This shows that by adhering to good laboratory practices and state of the art scientific procedures we can produce clones with few or no health issues.
In short, health problems in clones are not as serious as we might imagine, and even less so in dogs. As cloning techniques are optimized, these problems will tend to disappear.
The main causes of health problems in cloned animals are related to errors in highly complex biological processes such as reprogramming of the nuclei transferred during the cloning process and the imprinting of the embryos obtained.
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