Bringing the mammoth back from the dead

In a very interesting news feature in Nature, Henry Nicholls discusses the real possibilities of inverting the course of evolution by bringing the mammoth back to life.
During the last decades, hundreds of animal genomes have been published and the genome of the charismatic extinct Mamuthus primignenius has very recently joined the list.
Henry Nicholls’s paper summarizes the steps required to build up a mammoth from the genome to a living beast. He states that the whole process would involve: (1) “knowing exactly the sequences needed, (2) synthesizing a full set of chromosomes with the sequences, (3) transferring the nucleus into an egg and finally (4) transferring the egg into a womb…”
He says, none of the steps is currently possible.

 

Firstly, scientists would have to overtake the problem of DNA degradation which occurs in long-dead cells. High levels of degradation would create difficulties in assembling the DNA bases into a coherent sequence. Also, Nicholls points out that a very large sample size of individuals would need to be sequenced in order to achieve enough sequencing quality (allowing for corrections of errors occurred during DNA degradation). This level of quality (much higher than the quality required for research purposes) is necessary to actually create a “living being” from a genome sequence.

But even if good enough quality DNA sequences were obtained, there would be the succeeding problem of dividing the assembled sequences into chromosomes. Currently no mammoth cell has been  recovered with enough preservation quality to allow for chromosome counting. But even if one of these days a good cell is found, and scientists are able to go through the sequencing data to look for the beginning and endings of the chromosomes, this attempt will be complicated by the chromosome changes, gene deletions, duplications and arrangements that mammoths acquired since there diversion from their African ancestors. Still within this category of obstacles to building up a mammoth, the Y chromosome would pose even greater difficulties to assemble than the X chromosome (due to its repetitive nature). Some parts of the chromosomes are harder to assemble than others such as the centromeres and telomeres, but fortunately the use of artificial centromeres and telomeres is possible.

Another problem would be the fact that the genome data obtained so far provide only a single version of the sequences of the genome (1 set of chromosomes). If the mammoth was to be created with two identical chromosomes on each chromosome set, the expression of many recessive deleterious genes would be expected. Therefore much more sequencing work is required.

Nicholls goes on to explain that, after having all the necessary genome sequences at the required levels of quality, it will be the time to order them from a commercial DNA synthesis laboratory. Apparently, short strings of base pairs can be assembled into double-stranded DNA about 8000 base pairs long quite easily. However, as these stretches of DNA are stitched together, the DNA molecules become very unstable. Using the example of M. genitalium genome construction, the unstable stretches could then be put together into bacterial artificial chromosomes, in Escherichia coli. Then the larger stretches assembled in E. coli could subsequently be inserted into yeast artificial chromosomes, which are larger. However, scientists are aware that this procedure is unlikely to be used in reconstructing the mammoth genome, as the entire yeast genome is much smaller than a medium sized elephant’s chromosome. Nevertheless, there are hopes of development of chromosome reconstruction through new technologies, which aim at minimizing breaks, degradation and deletions that larger genomes are more prone to at this stage.

Next, Nicholls explains that once the chromosomes have been synthesized, they need to be put into a nucleus. That shouldn’t be too hard because since the 1980s that scientists know that naked DNA added to extracts of frog eggs quickly becomes wrapped up in proteins that condense it into chromatin. However, the challenge here would be to keep all DNA together to avoid miniature nuclei to be formed.

Uff….and there are more obstacles to overcome…is it really worth going on?
Ok, then…

After obtaining the nucleus with all the new chromosome constructs wrapped in, there will be more “mundane problems” to deal with. For example, collecting eggs from an elephant is rated as extremely hard due to their very short supply: the elephant’s oestrus cycle is a very long 16-week cycle, but ovulations are often skipped for five years or more due to gestation and lactation. Also the anatomy of the female elephant does not help: 1 metre of urogenital canal from the outside world to the hymen creates problems in reaching the ovocytes. 

The easiest way to reach the ovary, Nicholls reckons, would be to remove it from a deceased elephant and transplant it into mice with a suppressed immune system that would not reject them. During this process, the mouse oestrus cycle would have to be approximated to the elephant’s by removing its pituitary gland and using hormone treatments.

Now we have the egg available! Therefore, transferring the nucleus into the elephant’s eggs would seem possible, but again difficult steps would have to be taken to swerve around the obstacle posed by the elephant’s mitochondria (organelles that provide cells with energy through respiration), which have their own genomes specific to each species and therefore pose risks of incompatibility.  This means that artificial mitochondria would have to be built and inserted into the egg cytoplasm. Also the elephant’s mitochondria would have to be cleared out. 

Nucleus transfer technology is at the current time quite inefficient as few of the nucleus transfers resulting in embryos and many of the successful embryos give rise to life births with developmental abnormalities.

Nicholls explains that a better way to go from nucleus to embryo would be to use the stem cells produced by the new embryo (the result of the nucleus transfer into the egg elephant) even if it this embryo is doomed to abort. These stem cells could then be introduced in normal elephant embryos to create chimeras. In an elephant chimera, some cells are mammoth and some cells are elephants. Some of the chimeras would end up having mammoth cells in their ovaries and or testes. Apparently, gametes from germline chimeras have a much higher chance of producing successful embryos.

OK! Now we have an egg with a mammoth nucleus! Where is the mother?
Scientists reckon that, providing that a sperm-free component is injected into the womb to prepare the female’s uterus to receive the egg, this step would be relatively easier to achieve. Complications are not expected from size incompatibilities as most archaeological evidence indicates that that new born woolly mammoths were about the same size as newborn elephants.

Images

     The birth!

And finally in the after birth, scientists would be confronted with the need to create other specimens of both sexes. To make a species it is not enough to have an individual as enough diversity needs to be created to allow for their adaptability and evolution.
Also important, where would the mammoths live? A huge effort in ecosystem restoration would then need to be put in place.

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