In 1997 Dr Ian Wilmut and coworkers¹ startled the world by announcing the birth of Dolly, the first ever clone of an adult mammal (Figure 82.1). Because producing a clone of an adult mammal (i.e., a genetic replica of an existing individual) was thought biologically impossible, the announcement of Dolly’s birth reverberated around the world, capturing the attention of scientists and conjuring up fantastical visions from the lay public. Fascination with Dolly, continuing even today, stems from her unexpected origin. Dolly originated from the transfer of a quiescent-induced mammary cell originating from the udder of a 6-year-old adult ewe into an enucleated oocyte (i.e., an oocyte whose nuclear DNA had been previously removed). A brief electrical pulse was all that was needed to initiate embryonic development, highlighting the fact that the spermatozoon is not needed for this type of “asexual” reproduction.

The notion of cloning by transferring the nucleus of a cell into an enucleated oocyte was not novel to Dolly. The first successful production of clones by nuclear transfer was in amphibians in 1952.² Thirty one years later McGrath and Solter³ announced the first successful cloning of mice. Soon thereafter came the cloning of sheep,⁴, cattle,⁵ rabbits,⁶ and pigs.⁷ Interestingly, the early successes in obtaining cloned offspring were related to the use of totipotent or pluripotent cell types comprising the early embryo; any and every attempt using more differentiated cell types failed. Worldwide failures in different species gave credibility to the long and widely held dogma that it was biologically impossible to clone mammals by nuclear transfer. An initial hint that this dogma would not withstand the test of time came with the reporting of the births of Megan and Morag in 1996 by Dr Wilmut’s group with Dr Keith Campbell as lead author.⁸ Unlike the others that had been born up to that time, these cloned sheep were obtained by nuclear transfer using a cultured cell line that was embryonic in origin but which was passaged up to 6–13 times (i.e., number of times the cell line was subcultured for producing a larger number of cells from the initial number harvested). Because the cultured cell line was serum starved before nuclear transfer, the first ever success in obtaining live cloned offspring using a differentiated cell type was ascribed to the use of quiescent-induced cells. Subsequent successes in producing cloned sheep reinforced this notion by using established lines from mammary (i.e., Dolly), fetal and embryo cells after inducing cells into a quiescent state (Table 82.1).

Although the need for induction of quiescence before nuclear transfer was later questioned, Dolly’s birth served as a reminder to all that the impossible is possible assuming a willingness to persevere while thinking outside the box. The cloning of Dolly and the other sheep using cultured cell lines reinvigorated interests of scientists worldwide as the ability to do so provided the opportunity to genetically modify livestock species. Mindful that “the impossible was possible,” the list of adult animals cloned using cultured cell lines for nuclear transfer now includes, but is not limited to, sheep, cattle, mouse, goat, pig, gaur, mouflon, rabbit, cat, horse, rat, African wild cat, mule, banteng, deer, dog, ferret, wolf, buffalo, camel, and Spanish ibex (Figure 82.2). Realizing that what can be done in one species is likely possible in others, the list of different cloned species will continue to grow. Since Dolly, nuclear transfer clones have been constructed using a variety of different body cells such that cloning by nuclear transfer is now appropriately termed somatic cell nuclear transfer (SCNT).

Procedures involved in SCNT

The ultimate challenge of SCNT is to reprogram a somatic nucleus in a manner allowing for proper embryo development; in other words, transfer to an environment that would “force” the nucleus to forget its somatic programming and start functioning as a 1-cell zygote. The predominant cell type of choice for reprogramming a somatic nucleus is an oocyte arrested at metaphase II (MII). This is a logical choice for reprogramming a somatic nucleus because it has within it the majority, if not all, of the key critical components required for directing early embryo development. In general, SCNT is remarkably similar in the numerous species that have been cloned to date, highlighting the relevance of the procedural steps depicted in Figure 82.3.

Collection of somatic cells from the animal to be cloned (i.e., somatic cell donor) is a first important step in SCNT. In cattle and other species, adult animals have been cloned using a variety of different diploid (2n) cell types including, but not exclusive to, fibroblasts obtained from various sources, granulosa cells from antral follicles, cumulus cells, along with those originating from mammary tissue, muscle, oviduct, uterus and other sources. From a practical viewpoint, the relative ease of obtaining fibroblasts from a simple skin biopsy make this cell type a logical preference for cloning adult males and females. With regard to cows, ovarian/granulosa cells are easily obtained by the same ultrasound-guided transvaginal aspiration used for ovum pick-up (OPU). After collection somatic cells may be utilized immediately or after long-term culture. Dispersion into a single cell suspension is readily achieved using a trypsin-based solution with most somatic cell types. Interestingly, cell line rather than cell type is more influential on outcomes. In cases where outcomes are less than expected, individuals may choose to obtain another biopsy for establishing a different cell line from somatic cell donor before switching cell type. It is now known that induction of quiescence in somatic cells before nuclear transfer through serum starvation is not required for producing clones of adult animals.

The next important step in SCNT involves the collection of oocytes from donor females. Abattoir-derived ovaries provide an abundant and affordable supply of oocytes. Demonstrated ability to produce cloned offspring after in vitro maturation makes this the preferred source/approach for obtaining MII oocytes. In cases where it is important to control the genetic and/or maternal background of donor oocytes, OPU procedures provide a means for collecting smaller numbers of oocytes from live cows. In vitro maturation, while not as optimal as in vivo maturation occurring within Graafian follicle(s), provides a greater level of control for selecting MII oocytes after 18–20 hours.

The next and perhaps most labor-intensive step in SCNT involves the removal of maternal (nuclear) DNA from MII stage oocytes using microtools (see Figure 82.3). Addition of a microfilament inhibitor (i.e., cytochalasin B) to the holding medium relaxes the cytoplasm, allowing mechanical removal via aspiration of 5–15% of the oocyte’s cytoplasm containing the maternal (nuclear) DNA. Doing so is important to minimize the incidence of lysis that would otherwise occur without the addition of a microfilament inhibitor. Addition of Hoechst stain to the holding medium allows verification that the nuclear DNA was successfully removed. Limiting exposure to ultraviolet transillumination of the specific region of the microtool containing the maternal DNA avoids harm to the remaining “bag” of oocyte cytoplasm devoid of nuclear DNA but still containing mitochondrial DNA, essential organelles, and other essential components unique to the oocyte.

The next challenge of the SCNT procedure is transferring a somatic nucleus from a cell obtained from the animal to be cloned into the enucleated bag of oocyte cytoplasm. In most species, mice being an exception, this is achieved by electrical-induced fusion of the somatic cell with the enucleated bag of oocyte cytoplasm. To this end an intact somatic cell is mechanically inserted into the perivitelline space (available space between the zona pellucida and the enucleated bag of oocyte cytoplasm) using microtools. The resulting “couplet” (bag of enucleated oocyte cytoplasm and somatic cell) is aligned between two electrodes and briefly pulsed with an electrical current. In the case of cattle, 2.2 kV/cm for 40 s induces greater than 70% of couplets to fuse. Applying an electrical current induces pore formation in the respective membranes of the two cells. Electrofusion as a means of transferring the somatic nucleus into the bag of oocyte cytoplasm depends on continuing contact between the somatic cell and the oocyte cytoplasm. Depending on the extent to which membranes intermingle, fusion may occur. Within minutes of introducing the somatic nucleus into the bag of oocyte cytoplasm, the nuclear membrane breaks down and the chromatin condenses. Success in doing so effectively constructs the equivalent of a 1-cell embryo.

In some cases the electrical pulse utilized for fusion is also sufficient to “activate” the cloned embryo to begin development, while others choose various chemical combinations. Regardless of how one chooses to “jump start” embryo development, the ultimate challenge of activation protocols is to mimic the developmentally important processes occurring after fertilization. Note the deliberate use of the word “mimic” because the spermatozoon is not required for this type of asexual reproduction. After activation and depending on the species, cloned embryos may be transferred into ligated oviducts (preventing entry into the uterus of temporary recipients; this is what was done for Dolly), the uterus (pigs), or cultured in the incubator for a period of time required for development to the compact morula or blastocyst stage (cattle). Cloned bovine embryos develop to the blastocyst stage at an equivalent rate as those undergoing in vitro fertilization (Table 82.2).

Embryo transfer into surrogate recipients uses the same approach as for transferring in vitro– or in vivo-derived embryos, but with a few extra challenges. Depending on the level of difficulty, cloned embryos may be damaged while being transferred into the uterus of the surrogate recipients. In cattle, effort should be taken to transfer embryos before or during the early stages of blastocoele expansion, otherwise the expanding blastocoele will protrude through the holes in the zona pellucida created by earlier use of microtools (Figure 82.4). Extensive protrusions appearing as a “figure 8” could explain why twins are occasionally noted after the transfer of individual cloned embryos. Pregnancy rates after the transfer of one or two embryos do not differ. Transfer of a single embryo is helpful for minimizing complications associated with twinning. Nonetheless, earlier stage embryos may be transferred (i.e., compact morula or early blastocyst stage embryos) to avoid possible issues with the aberrant hatching of clones. When doing so, extra care should be taken to ensure that embryos are stage-matched with the uterus (i.e., compact morulae would be transferred to day-6 recipients, not day 7 or 8). Depending on the cell line, it is reasonable to expect day-28 pregnancy rates in cattle approaching 50% after the transfer of single embryos, as indicated by the presence of an embryo proper with heartbeat (Table 82.3). A limited number of cloned embryos resulting in a confirmed pregnancy will progress to term and result in the delivery of live offspring.