Organismal Cloning—Advent of Stem Cell and Transgenic Animal Biotechnologies
As the field of modern genetics blossomed in the 1950s, the prospect of cloning whole organisms became both appealing and possible. Organismal cloning can be achieved by three primary means: (1) the splitting of a single embryo into two identical embryos,19 (2) the fusion of an embryonic cell with an unfertilized and enucleated egg, or (3) the transfer of all nuclear contents from an adult or embryonic somatic cell to an unfertilized, enucleated egg (see Figure 1.1). The latter, also called somatic20 cell nuclear transfer (SCNT), is the most powerful technique. In either embryonic fusion or SCNT, the recipient egg is reactivated, and grown in vitro to form a multicellular embryo. A cloned embryo is implanted into a surrogate mother, or used for medicinal purposes.
Figure 1.1. Forms of organismal cloning.
When the final product of the cloning procedure is the embryo, or the stem cells that can be grown from it, the process is called therapeutic cloning (see Figure 1.1). Stem cells are undifferentiated cells that play critical roles in growth, development, and tissue repair throughout the body. Stem cells may differentiate into new, more specialized, cell types. When this occurs they self-renew by asymmetrical cell division. One daughter cell remains a stem cell and the other differentiates into a specific cell type (e.g., nerve, skin, lung). The elasticity of a stem cell is described in terms of potency. Stem cells that are the progenitors of many cell types are pluripotent.21 Pluripotent stem cells like those found in embryos are the holy grail of regenerative medicine because they can be used to study rare diseases, heal wounds, replace dying cells, or even grow replacement organs. The biology of stem cells is discussed in detail in Chapter 2.
When an embryo derived by SCNT is implanted into a surrogate mother for further development into a whole organism, it is called reproductive cloning (Niemann and Lucas-Hahn 2012). The cloned animal that results is genetically identical to the donor nucleus from which it is derived. Despite this, there are differences between the cloned and original animal. Most notably, the two are age-matched, due to asynchronous gestation periods.
The first successful SCNT was accomplished in 1952 by Robert Briggs and Thomas King (Gurdon 1997). They produced a cloned tadpole by replacing the nucleus of a leopard frog egg with that of an early-stage embryo (Gurdon 1997). In their work to follow, they noted two things: many cloned embryos developed abnormally, and the more differentiated (i.e., mature) the donor nucleus, the less successful the cloning (Gurdon 1997).
The technology progressed incrementally from there, with organisms such as rabbit (in 1975), mouse (in 1981 and 1983), sheep (in 1986), pigs (in 1989), and cow (in 1994) (reviewed in (Gurdon and Byrne 2003)). Along the way, based on the findings of Briggs and King, researchers assumed that viable offspring from reproductive cloning could only be achieved using donor nuclei from early embryonic cells. This assumption was debunked in 1997, with the birth of Dolly,22 the cloned sheep (Wilmut et al. 1997). What made Dolly so special was that her donor nucleus came from the mammary gland of an adult ewe (Wilmut et al. 1997). That is, adult organisms could—for the first time—be cloned. This accomplishment was highly publicized and sparked a widespread debate pertaining to the ethics and implications of organismal cloning.
Not long after the birth of Dolly, organismal cloning took another leap when the first GM animal clone was born (Schnieke et al. 1997).
Box 1.1. Misconceptions about organismal cloning
- • Organismal cloning is a new technology.23
- • Clones are always artificial.24
- • All clones are abnormal and die young.25
- • Animal cloning is rare.26
- • Milk and meat from cloned animals is excluded from the U.S. food supply by law.27
- • The first-ever animal clone was Dolly the sheep.28
- • A clone is the same age as the original organism.29
- • Clones are exactly identical in every way.30
- • The same procedure is used to clone all animal species.31
- • Human cloning is prohibited by international law.32
- • Extinct animals, such as mammoths or dinosaurs, have been
This lamb—named “Polly”—was created from the nucleus of a tissue culture cell in which the human Factor IX gene was inserted. As a result, Polly produced human Factor IX, a blood clotting protein, in her milk.34 A 2002 report in Science announced the production of pig clones that had been modified to express human-like cell surface markers (Lai et al. 2002). Xenotransplantation (i.e., tissue transplantation between species) is a potential medical application of such humanized animals. In 2003, GM and cloned dairy cows were created. These cows produce milk with enhanced protein content (Brophy et al. 2003). Cloned goats, genetically modified to produce spider silk proteins in their milk, captured headlines and imaginations in the early 2000s. The goal of “silk milk” goats was the large-scale harvest of spider silk for the production of extraordinarily strong, elastic, and lightweight textiles35 (Majumder, Kaulaskar, and Neogi 2015). This “BioSTEEL” textile could be used for bulletproof clothing or surgical sutures.
With over a dozen animal species cloned since the late 1990s (Verma et al. 2015), what about primates? As early as 1997, rhesus monkey embryos were successfully cloned by nuclear transfer from blastomeres36 into enucleated eggs (Meng et al. 1997). Two live clones were born— Ditto and Neti—as a result of three pregnancies and 29 implanted embryos (Meng et al. 1997). While the birth of live nonhuman primate clones made by adult cell SCNT has not yet been actualized, multiple pregnancies have been reported (Sparman, Tachibana, and Mitalipov 2010).
It is generally accepted that human reproductive cloning has not been done, although there is no international or U.S. federal law prohibiting it.
In 2005 the United Nations adopted a Declaration on Human Cloning, in which members agreed to prohibit any human cloning that was “incompatible with human dignity and the protection of human life” (Nations 2005). Member nations have been unable to come to a consensus on what exactly these conditions mean in practical terms (Nations 2005). In the United States, reproductive and therapeutic human cloning are regulated at the state level. The 13 states that currently ban human reproductive cloning include Arkansas, California, Connecticut, Iowa, Indiana, Massachusetts, Maryland, Michigan, North Dakota, New Jersey, Rhode Island, South Dakota, and Virginia (Ayala 2015). In other states, private—but no public—funding may be used for research involving human cloning or human stem cell lines established after August 9, 2001 (Radio 2016; Ayala 2015).
The history of human cloning is complicated by veiled, falsified, and unsubstantiated claims. In 1999, Advanced Cell Technology (ACT), a leading biotech company at the time, announced the creation of the first cloned human-cow hybrid embryos (News 1999). The details of this landmark experiment remain unclear, but it is thought that the hybrid embryos were made by transferring human donor nuclei into enucleated cow oocytes37 (News 1999). ACT allowed the hybrid embryos to grow for 12 days before they were destroyed (News 1999). In 2002, CLONAID—a company associated with a religious group that believes humans were created by extraterrestrials—announced the birth of the first cloned human, whom they named Eve. Since then, the company has advertised human cloning services to overcome infertility (Clonaid 2006-2009). None of CLONAID’s claims have been substantiated by the scientific community (Institute 2016a).
In 2004, and years ahead of others in the field, Woo-Suk Hwang’s lab at Seoul National University reported the development of 11 human embryonic stem cell lines from the SCNT of adult donor nuclei (Hwang et al. 2004; Hwang et al. 2005). Not long after, some of Dr. Hwang’s junior colleagues accused him of falsifying data and unethically obtaining human egg donations (Sang-Hun 2014). Following an investigation, Science retracted his articles in 2006. He was later convicted of embezzling research funds and bioethical misconduct (Sang-Hun 2014). Hwang was dismissed from Seoul National University, but continues to perform animal cloning. In 2005 he announced the creation of the first cloned dog, Snuppy (Sang-Hun 2014). He now oversees Sooam Biotech Research Foundation, a company in South Korea that offers cloning services to pet owners.38
Real advances in primate cloning have come more gradually. In 2007, Byrne et al. created the first primate embryonic stem cells (i.e., rhesus macaque) derived from the SCNT of adult fibroblasts (Byrne et al. 2007).
This work was a pivotal proof-of-principle for the development of patient- matched embryonic stem cells. In 2008 French et al. went further by creating human blastocysts by SCNT using adult donor nuclei (French et al. 2008). And finally in 2013, the first confirmed patient-matched human embryonic stem cells were made using adult fibroblast cells as nuclear donors (Tachibana et al. 2013). Collectively, this work holds immense promise for the development of personalized embryonic stem cell therapies for human patients.
While the primate therapeutic cloning technology advanced, so did the equally promising field of stem cell reprogramming. In 2006, Yamanaka and colleagues were the first to transform—or reprogram—adult mouse cells into those relatively indistinguishable from embryonic stem cells (Takahashi and Yamanaka 2006). They did so through the expression of what came to be known as the Yamanaka factors—transcription factors Oct4, Sox2, Klf4, and Myc. These transcription factors regulate genes that cause cells to undergo a sort of amnesia in which they forget their cellular identity (Takahashi and Yamanaka 2006). The new cells that Yamanka et al. created by introducing and expressing the Yamanka factors were called induced pluripotent stem cells (iPSs) (Takahashi and Yamanaka 2006). As little as two years later, human iPSs were created by a similar procedure (Park et al. 2008). Since then, iPSs have been successfully reared into a variety of cell types and even used to populate bioscaffolds for the growth of whole organs. The iPS technology bypasses controversial human embryo creation and destruction in the production of malleable patient-matched stem cells for regenerative medicine.