Ian Wilmut Ph.D.
Jane Taylor University of Edinburgh
Besides Sheep and Cattle, Cat, Deer, Dog, Horse, Mule, Ox, Rabbit, Rat, and Monkey Have Been Cloned from Somatic Cells
A new era was heralded by the birth of Dolly, born in July 1996 after transfer of the nucleus from a mammary cell of an adult ewe to an enucleated oocyte. In this editorial review, we describe the research that led to the birth of Dolly and discuss some of the implications of the Dolly experiment in changing the way we think about cell plasticity.
Research That Led to the Birth of Dolly
Dolly was the first animal to be born following transfer of a nucleus from an adult donor. Although this success followed research over several decades in amphibians and laboratory mammals, it followed specifically from the recognition of the need to co-ordinate the cell cycles of donor nucleus and recipient oocyte. There had been recognition of the importance of this relationship, but the work of Keith Campbell at Roslin provided the first detailed description of the changes in level of regulatory molecules (meiosis/mitosis promoting factor) in the mammalian oocyte and the early embryo after fertilization, and the effect on transferred nuclei.
This research suggested two protocols for nuclear transfer in sheep that are expected to produce embryos with normal ploidy. In one, the oocyte is activated before enucleation and transfer of a nucleus, which may be at any stage of the cycle. In this case, the transferred nucleus determines whether or not DNA replication occurs. In the second protocol, nuclei that are awaiting DNA replication are transferred into oocytes in metaphase II of meiosis. In this case, DNA replication occurs in an appropriate manner because the transferred nucleus is awaiting replication and the cytoplast is primed to carry out the replication. All other combinations are expected to lead to errors of one kind or another and have to have limited developmental potential.
Optimization of the Roslin Procedures
This initial analysis defined procedures that yield embryos with normal ploidy. Further research demonstrated that a greater proportion of reconstructed embryos developed to term if they were produced by transfer of a nucleus in G0/G1 to an oocyte in metaphase II. This difference is thought to reflect the greater efficiency with which the oocyte cytoplasm reprograms gene expression of the transferred somatic cell nucleus to that of an early embryo.
Production of embryonic cells in G1-phase is time-consuming and not entirely accurate. This is particularly the case for embryo-derived cells, which do not respond to the normal checkpoints. Keith Campbell introduced the use of cultured cells in G0, through which tissue-derived cells exit the cell cycle and become quiescent if cultured in a low concentration of serum. In this case, the quiescent cells are in a stable state and can be left in an incubator or on the bench for as long as nuclei are required for transfer. This innovation facilitated nuclear transfer and yielded more reproducible results.
Modifications to the Roslin Protocol
These analyses led to the birth of Dolly and provided the biological basis for research in other species. In most species, viable offspring were obtained using the Roslin procedure. Primates and rats were two exceptions.
When the Roslin procedure was applied in human and nonhuman primates, development was limited to the blastocyst stage until it was discovered that handling and enucleation of primate oocytes according to existing procedures induced partial activation of the oocyte. It was noted that under these circumstances, the activation procedure applied after nuclear transfer was unable to induce a full response in the oocyte and development of reconstructed embryos was limited.
Procedures for handling and enucleation had been modified so as not to induce premature activation. It is argued by some practitioners that human embryo stem cell lines from cloned embryos offer the best possible cells for use in cell therapy. In particular, this is because they would be genetically identical to the patient, if the nuclear donor cell is taken from the patient. This approach contrasts with that of using iPS cells that are homozygous at major HLA loci to provide useful partial match to recipients.
Progress in the rat was hindered by technical limitations such as poor-quality culture systems that failed to culture embryos beyond a two- to four-cell block, and problems with spontaneous activation of oocytes on removal from the oviduct. These issues were overcome through modifications to the culture medium and the use of a proteasome inhibitor (MG132) to stabilize the oocytes at metaphase II. Somatic nuclear transfer in the rat remains problematic, since the publication in 2003 describing the birth of two viable offspring using the Honolulu mouse protocol, subsequent publications have failed to report births of live cloned rat pups.
Precise Genetic Modification of Livestock
The cloning project at Roslin Institute was initiated with the aim of being able to introduce precise genetic modification into livestock. This would be achieved by introduction of the desired change into nuclei from a donor with a suitable background. After confirmation of the desired genetic change, these cells would be used for nuclear transfer. The resulting offspring would have the characteristics of the selected animal with the additional, selected modification. As a demonstration, the group at Roslin used this approach to introduce sequences in which regulatory sequences from CASEIN/BETA lactoglobulin directed production of human clotting factor IX into the milk of sheep.
The Roslin procedure for nuclear transfer has been used by others to introduce genetic change, for example, to create models of the human genetic disease cystic fibrosis in pigs. While this protocol was effective, a more efficient alternative procedure for the introduction of precise genetic changes has been provided by the development of the “clustered, regularly interspaced, short palindromic repeat” (CRISPR) technology described by Sander and Joung. These systems are so efficient and accurate that it is apparently possible to simultaneously modify several genes in mouse embryos using the CRISPR/Cas9 system, and with optimization this may also be possible in livestock species
To read this article in its entirety please visit “Cloning After Dolly” as published in Cellular Reprogramming.
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