A clone is a population of cells or organisms derived asexually from a single ancestor. They are genetically identical to each other to their common ancestor. Cloning means the production of exact genetic replica copies of an individual. They can not be considered as an offspring but simply the copy of a given individual. Much work has been done on cloning in plants and microorganisms. However, the techniques used in plants can not be applied for animals. Moreover, many animals from a single genetically superior embryo can be produced. Still there is no method of finding out which embryos are capable of cloning. It is useless to clone an embryo if it is not superior.
During 1960s, Beatrice Mintz at Cancer Research Institute, Philadelphia (USA) demonstrated the interesting experimentation. She carried out fusion of embryos of two different species of mouse. This resulted in the formation of a single embryo which finally developed into a normal healthy animal having four parents. Embryo A was derived from the cross of male x female of one species, and embryo B derived after cross of male x female of the other species. The embryo A and B were united together and produced a single mass. In this experiment Mintz removed zona pellucida membrane of two early embryos and placed them in a suitable culture medium. The embryonic cells of the two embryos of blastula stage united randomly into a single mass of double sized embryo (blastocyst). A fresh membrane developed around the embryo. Then the embryo was transferred into the uterus of a foster mother. The foster mother was mated with a sterile male to bring her into proper stage for implantation. The first offspring with four parents was born in 1965. Similarly, exciting experiments have been done on another mammal. Following the same technique a hybrid of goat and sheep named geep was produced (Joshi, 1998).
At present two types of techniques for embryo cloning viz., nuclear transplantation (transfer) and embryonic stem cells, are being developed.
Fig. 7.3. Nuclear transplantation in frog (diagrammatic).
Nuclear transplantation (also nuclear transfer) involves removal of a single blastomere from a cleavage stage embryo with a fine micropipette of glass, and placing it under the outer membrane of an unfertilized mature enucleated oocyte (whose haploid nucleus has been removed by using micropipette or destroyed by UV light). For the first time in 1955, Robert Briggs and Tom King at Cancer Research Institute, Philadelphia (USA) carried out nuclear transplantation experiment on embryonic cells of frog. They transferred nucleus of undifferentiated blastula (a stage soon after fertilization of egg) into an enucleated egg cell. They noticed the normal development of the embryo. When they performed serial transplantation of differentiated nucleus from late gastrula (a stage after blastula) into a nucleus-free unfertilized egg, abnormal embryos were formed. This shows that cell nucleus is differentiated with embryo development. In 1960s, J.B. Gurdon at Oxford University, U.K. transferred differentiated intestinal nucleus of a frog into nucleus-free unfertilized egg of different amphibian species (Xenopus laevis).
The embryo developed into tadpole and matured into frog (Gurdon, 1962). This new enucleated cell developed into normal embryo. Any damage to the donor nucleus during transplantation leads to abnormal development (Fig. 7.3).
DOLLY - The first mammalian clone. 'Dolly', the worlds' first mammalian clone has been created from a fully differentiated non-reproductive cell of an adult sheep. It was born in February, 1996. The name Dolly has been given after an American country singer, Dolly Parton. In 1995, Ian Wilmut and his team of researchers at Roslin Institute, Edinburgh, Scotland, took udder (a fully differentiated tissue) from six year old sheep, Fin Dorset Ewe, and placed it in special solution that controlled cell cycle of cell division. The cell was deprived off certain nutrient. At the same time an unfertilized egg was obtained from another adult sheep (Fig. 7.4). Its nucleus was carefully removed leaving the intact cytoplasm in egg. The nucleus of udder cell was taken out and transferred into nucleus-free egg. This was facilitated by applying mild electric sock. The newly transplanted nucleus soon became functional according to the new cytoplasm in which it had been artificially transferred. This viable combination underwent cleavage like normal zygote. This so called embryo was then transplanted into the uterus of a third adult sheep (surrogate mother/foster mother) for its further development.
Fig. 7.4. Wilmut's cloning experiment showing the birth of Dolly through nuclear transplantation technique.
Finally, a normal healthy little lamb, Dolly was born in February, 1996 which was genetically similar to the clone mother
from which nuclear DNA was taken out. It does not have any similarity with that sheep from which egg was taken out or surrogate mother because they did not contribute any genetic character (Wimut et al.,
1997). Thus, Dolly has only a single parent because she has born asexually, a characteristic feature found in lower forms of animal life, not in mammals.
Although behind this great success the rate of success is very slow, yet it has given some hope to embryo-biotechnologists to bring about refinement. Out of 277 nuclei transferred singly to enucleated egg, only 29 eggs grew into embryos. Out of these, only 13 embryo could be successfully transplanted into surrogate mothers. Of these only one ewe was successful in giving birth to an offspring, Dolly (Wilmut et al,
The significant considerations that can be derived from this experiment are that: (i)
the genes of differentiated cells have inherent totipotency, (ii)
the interplay between the regulatory system of the genome in nucleus and the cytoplasmic factors of egg may make a cell totipotent, (iii)
possibly the cytoplasm of enucleated egg makes the transplanted nucleus totipotent like that of normal fertilized egg nucleus, (iv)
the maternally derived information in egg cytoplasm has an important role in cleavage which usually occurs after fertilization.
Fig. 7.5. Production of chimeric mouse by embryonic stem cell transplantation.
Hence, it is the egg cytoplasm but not the nucleus that regulates cleavage. Because the udder cell nucleus has limited potential for mitosis; it is the egg cytoplasm that interacted intracellularly with udder cell nucleus and stimulated to undergo repeated mitosis, (v)
carbon copy of the adult sheep could be produced without involving sperms from male partner, and (vi)
the cloned animal produced via nuclear transplantation technique will be capable of restoring fertility as in 1998 Dolly gave birth to a little lamb named Bonny.
Embryonic Stem (ES) cells
Cloning of mice could not be done as in sheep via nuclear transplantation. This was due to acceleration of developmental programmes of embryo. However, it is evident that before first embryonic division the cell has started its process of differentiation. Therefore, for cloning of mice an alternative approach has been made i.e.
the use of ES cells. A blastocyst of mouse is place in culture condition. The inner cells that form future foetus continue to divide and remain in undifferentiated totipotent state as ES cells. There is a peptide growth factor known as leukaemia inhibitory factor (LIF) which establishes and maintains ES cell lines. The ES cell lines will be very useful in the area of production of transgenic animals (see
preceding sections). However, the ES cells are used in two different ways: a small number of ES cells can be injected into blastocoel space of a blastocyst (Fig. 7.5). The ES cells get mixed with inner mass of cells of blastocyst to produce a chimera mouse which is a mixture of two cell genotype having the patches of different colored fur. Crossing of male and female chimera will allow selection of homozygous mice derived from ES cells (Read and Smith, 1996).