How cloning works

Shoukhrat Mitalipov says cloning human cells was like winning a lottery, but his success has nothing to do with luck. – Dieter Egli, NYSCF

Ian Wilmot cloned Dolly the sheep in 1996. It took until 2013 for Shoukhrat Mitalipov to repeat the feat in a human being. What did he do differently? “It was like getting all the right lottery numbers in row,” says Mitalipov. But his success had nothing to do with luck – it took 17 years of methodical trial and error to optimise every step.

Cloning involves replacing the DNA of an egg with the nucleus of a donor cell. Factors in the egg reboot the donor’s nucleus, to a stage where it can direct the development of a new embryo. But in cloning, everything is about choreography – and every species, it seems, does the dance a little differently.

Most of the optimisation was carried out in the rhesus monkey. First off, Mitalipov found a problem with the standard method of sucking out the egg’s own DNA, so-called “enucleation”. It involved making the egg DNA visible with a Hoechst dye and viewing it under UV light, both highly damaging to the egg. Mitalipov developed a more benign microscopic imaging technique.

Second, eggs exist in a resting state known as “meiotic arrest”. It was very important not to rouse them too early. The Dolly method inserted the entire donor cell just below the shell of the egg, abutting the egg’s outer membrane. Then he applied a local electric shock that fused the two cell membranes, enclosing the donor nucleus within the egg. The problem was, in human cells the electric shock also jolted the egg out of its arrest too early. Mitalipov’s answer was to use a virus to fuse the cells instead, coating the donor cell in Sendai virus before inserting it under the egg shell. He also bathed the egg in a cocktail consisting of caffeine to keep the egg under arrest during this time. Finally it was time to release the egg from its “arrest”.

Every step of the process relies on the skill of the operator.

Normally this go signal is given by the sperm’s entry. An electric pulse and chemicals (protein synthesis and phosphorylation inhibitors) given at this stage was crucial to the success of the technique in humans. Others had got this far; the egg, promisingly, would divide a few times but then grind to a halt. The problem was the donor genome had not properly rebooted. To spur it on he used “HDAC inhibitors”, chemicals known to activate dormant DNA.

Finally the egg divided to the 128-cell blastocyst stage. Inside was the inner cell mass that would be removed and cultivated to provide embryonic stem cells.

Every step of the process relies on the skill of the operator. Mitalipov’s lab was able to achieve the extraordinary result of deriving four embryonic stem cell lines from eight eggs. Overall the quality of the starting egg is very important.

Mitalipov is delighted that two labs have now replicated the method with older donor cells; his own lab has also repeated the feat with cells from a 72-year-old donor with amyotrophic lateral sclerosis. “For 17 years no one could do it; now in six months they can.”

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