MULTIPLE GENETICALLY IDENTICAL. FROGS. J. B. GURDON*. IT IS often desirable to conduct bio- logical experiments with genetically identical animals.
MULTIPLE GENETICALLY IDENTICAL FROGS J. B . GURDON*
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Method Nuclear transplantation is in effect a means of replacing the zygote nucleus of a fertilized egg by an embryonic cell nucleus. The principle of the method is shown diagrammatically in Figure 1; it consists of dissociating embryonic cells, and then injecting a single cell, the wall of which has been broken, into an unfertilized and enucleated egg (Briggs and King 1 ). In its application to the experiments with Xenopus, the tech-
nique has been modified in various ways. Recipient eggs are enucleated by exposing their animal pole to ultraviolet irradiation; this kills the egg nucleus, but does not damage the egg cytoplasm5. Use is made of a nuclear marker which gives proof that the nuclei of transplantembryos are derived from the transplanted nucleus and not from the recipient egg nucleus (Elsdale et al.3). In the case of Xenopus, such transplantembryos have been reared into adult, sexually mature frogs capable of normal reproduction (Gurdon et al.s). Recent evidence from the results of nuclear transplantation experiments has provided reasons for believing that cell nuclei come to differ from each other in the course of embryonic differentiation, and that they are therefore only identical up to a certain stage in development (Briggs and King 2 , Fischberg et al.*). An analysis of the potentialities of endoderm nuclei in Xenopus indicates that nuclear differentiation does not begin in this species until after gastrulation (Gurdon 7 ). Thus in Xenopus all transplant-embryos or frogs derived by transplantation of endoderm nuclei from a single blastula or gastrula should be genetically identical. Additional frogs, genetically identical to these, can be obtained by serial nuclear transplantation; this involves exactly the same technique as for first transfers, but donor nuclei are in this case taken from a blastula or gastrula embryo which has itself been derived from a transplanted nucleus. Thus the only difference between direct and serial transplantation is that donor embryos from fertilized eggs are used for direct or first transfers, but embryos derived from transplanted nuclei for serial transfers.
*Beit Memorial Research Fellow. Department of Zoology, Parks Road, Oxford, England. The author is most grateful to Dr. M. Fischberg for having pointed out the value of genetically identical frogs, and for his help with this manuscript.
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T IS often desirable to conduct biological experiments with genetically identical animals. These are easily obtained in organisms which can be reproduced vegetatively like most plants and micro-organisms, and in many invertebrate animals. Vertebrates, however, only reproduce by sexual means, and so give rise to offspring which differ from each other in some respects at least. Identical twins sometimes arise naturally, but it is unusual for more than two such individuals to be obtained. The progeny of entirely homozygous parents would be genetically identical, but although a certain degree of homozygosity can be obtained by several generations of inbreeding, it apparently cannot be much increased by prolonging the process. Apart from this difficulty, homozygosity tends to be associated with weakness, physical abnormality, and sterility. It is therefore very desirable that a method should be available for obtaining many genetically identical individuals among the vertebrates. Such individuals have been produced in the case of the frog, Xenopus laevis, by making use of the technique of nuclear transplantation.
The Journal of Heredity
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Figure 2
The upper two rows show adult male frogs derived from transplanted blastula nuclei of Xenopus laevis, and from recipient eggs laid by one frog. The controls (bottom row) are derived from fertilized eggs of the same parentage as the blastula donor which provided the nuclei for the 10 transplant-frogs above. The controls consist of adult males and females. The pronounced cloaca typical of females can be seen in the second frog from the left on the bottom row.
This process of serial transplantation, illustrated in Figure 1, may be continued indefinitely, giving rise to genetically identical individuals which need be limited in number only by the industry of the operator.
Factors Affecting Similarities Among Genetically Identical Frogs Frogs obtained by direct and serial transplantation of blastula or gastrula nuclei all have an identical genotype. A consequence of the nuclear transplantation technique is that frogs may be obtained from identical egg cytoplasm as well as from identical nuclei. This is the case when nuclei are transplanted into recipient eggs laid by the same frog. In the case of serial transplantation, recipient eggs have to be obtained from different frogs for each transfer genera-
tion, since the laying period of each frog is limited in duration; however, no case has yet been observed in experiments with Xenopus laevis when frogs with identical nuclei differ according to the origin of the recipient eggs. Genetically identical frogs, obtained by first, second, third and fourth transfers of nuclei, were indistinguishable from each other although recipient eggs from a different female were used for each transfer generation (Gurdon 8 ). In spite of the innate identity of the donor nuclei and recipient eggs, there are, nevertheless, certain factors which may be responsible for phenotypic differences among genetically identical frogs. One concerns possible damage to the donor nuclei or recipient eggs during the transplantation procedure. The effects of inconsistencies in carrying out
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TRANSPLANTS AND CONTROLS
Gurdon: Genetically Identical Frogs
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