The Message From Artificial Clones

Nature-Nurture-Noise (7)

Not that long ago, the notion of human-manufactured clones was confined to Science Fiction. Now it is commonplace. It began with frogs as a proof of concept. But the initial motivation for developing the technology was to replicate desirable traits in domestic mammals without the hassle of actual breeding, or even artificial insemination. These human-manufactured mammal clones provide another avenue to investigate the random component in individuality.

The most common method of cloning is called somatic cell nuclear transfer (SCNT). A somatic cell is any cell other than sperm or egg. Skins cells, for example. The nucleus of the skin cell is removed (called enucleation) then emplaced in an egg. But before the skin cell nucleus is transferred to the egg, the nucleus already in the egg must be removed. Because the egg cell is unfertilized it contains only half of a genome, that of the female that produced it. Because the whole genome is transferred there is no need for the egg too be fertilized; it can be immediately implanted into the uterus of the surrogate mother.

The first mammal clone to be successfully produced by this method was a sheep named Dolly, after Dolly Parton, a droll reference to the source of the nucleus, a cell from a mammary gland, the source of milk, which resides in human breasts and sheep udders. (For those who may be unfamiliar with Dolly Parton, her breasts are famously capacious.) Dolly, the sheep, was very lucky. The vast majority for SCMT clones fail to develop much at all, most die before birth, most of the rest die soon after birth, most of those that survive for any length of time are obviously compromised in fundamental physiological ways. Dolly herself had a short lifespan. There are several reasons for the inefficiency of this cloning process, aside from the technical and technological.

In general, the failures stem from a naïve genetic determinism, that there is a genetic program in the genome that precisely determines the course of development, independently of any input from all the other stuff that makes a cell a cell. What should have been apparent then is blindingly obvious now: there’s a lot of other developmentally relevant molecules in the cell in addition to DNA. The egg cell is particularly rife with these non-genetic determinants. In fact, the earliest developmental events occur completely independently of the embryonic genome. Among these non-genomic actors are maternal factors in the cytoplasm—the stuff outside the nucleus—of the egg cell; these include various proteins and RNAs from the mother that influence early, formative epigenetic events. It is pathologies in these early epigenetic processes that are responsible for most failed clones, as I have discussed in more detail elsewhere.

After Dolly, animal cloning rapidly accelerated. More sheep, then cattle, pigs, horses, goats, and camels. Well over 1,000 dog clones have been produced. Many of the dog clones were generated for laboratory research but some were attempts to replicate beloved pets. The first beloved pet to be cloned, however, was a cat. This cloned cat is particularly instructive for our purposes DOI: 10.1126/science.295.5559.1443a.

The cat clone was named CC (for Copy Cat, some say Carbon Copy), which proved more aspirational than apt. The source (mother?) of carbon copy was a female calico, a particularly unfortunate choice for copying purposes. Unfortunate because it was a female, for reasons to be discussed in a future post, unfortunate also because it was a calico, a color pattern impossible to replicate through cloning, for reasons also to be discussed in a future post. Carbon copy was a grey tabby, without a hint of the brown and orangish-brown hues that distinguish calico (and tortoiseshell) cats from the merely two-toned. That this came as a surprise is testimony to the naïve genetic determinism that motivated the project, but also a naïve determinism more generally. Randomness played a huge role in rendering Carbon Copy so discrepant. This should have been anticipated given Sewall Wright’s research on the inheritance of coat colors in guinea pigs almost a century earlier.

Recall that the color pattern of particular interest to Wright was piebald (black splotches on white background). Most domesticated mammals include piebald breeds. Of these, Holstein/Friesan cattle have been a particular target for cloning because of their economic importance. The most obvious way in which Holstein clones differ from their source is in the distribution of black and white patches, but also in the amount of white versus black surface area.

First, it is important to note that white is not a color, it is the absence of color. The white areas are depigmented. The black areas, on the other hand, are enriched in melanin, the pigment for which melanoma is named. The ultimate source of melanin are neural crest cells, which originate early in development but then undergo a long migration from their embryonic source to the target tissues. Though coordinated to a high degree of precision, there are always random perturbations in the journey, especially as the neural crest cells near their targets. These perturbations are responsible for the deviations—within clones—in the distribution of white, depigmented patches over the black background. A particular piebald pattern in any mammal is as replicable as a fingerprint. Add another pigment to the mix, as in calico cats and things only get worse, for those who want their copies to look like actual copies.

Another source of variation within cattle clones is in the conformation of their horns. Holsteins and other dairy cattle are dehorned as calves, so we must look elsewhere to appreciate clonal variation in this trait. One breed that doesn’t get dehorned is the Korean Hanwoo. This breed has garnered a lot of attention in the cloning community. Originally bred for physical labor (draft animals), the Hanwoo almost became extinct when more efficient mechanical alternatives were introduced. Fortunately for the breed, unfortunately for the individual Hanwoos, it was noticed that their meat was particularly delectable, especially when they were spared physical labor. Today, Hanwoo meat is the most favored by foodies, even more so than Wagyu, a Japanese breed that is derived from the Hanwoo. (Kobe beef is Wagyu raised near the city of that name.)

How better to create even more delectable Hanwoo beef than to clone the very best Hanwoo cattle? Whether the muscle chemistry was precisely replicable is unknown, but the horns were much less replicable than you might expect. Of three clones derived from one genome, one individual had horns that closely resembled those of the “mother”, another, though, had horns that pointed downward, still another’s horns were completely asymmetrical, the horn on one side taking a turn toward the horizontal.

Less surprising was the variation in nose prints. For cattle noses, like human noses, fingers, and palms, have dermal ridges. No two individual Hanwoo have the same dermal ridge pattern on their noses, including, it turns out, Hanwoo clones. Nose prints in cattle have the same potential forensic value as human fingerprints. The same goes, presumably, for all mammal species with sufficient terminal nose surface area.

Pigs and goats have also been extensively cloned. Among the most obvious differences among cloned pig littermates are patterns of hair density. This is especially noticeable along the spine; some clones have very densely haired backbones, in others the backbone hairs are sparse. But it is in their differences in total body size that clone mates most strikingly differ. Size differences are present at birth but increase in the juvenile phase, even when fed in an identical manner. Other traits that differ markedly in clonal pigs include blood calcium, glucose, and albumin levels. Compared to their outbred counterparts, some of these traits are more variable in the clones 10.1095/biolreprod.103.016147.

Goat clones also vary in size as measured nearly a year after weaning. In one study, some of the hormones relevant to growth—including Insulin-like Growth Factor and Thyroxin—were also measured, and these too evidenced large individual differences https://doi.org/10.1089/clo.2005.7.214. The hormone most relevant to growth is unsurprisingly called Growth Hormone. Individual differences in the levels of this hormone among individuals with identical genomes is truly astounding. Even in the face of a technology designed to create uniformity, through genetic manipulation, Individuality will out!