Gurdon JB et al. (1975), The developmental capacity of nuclei transplant...

XB-ART-32706

J Embryol Exp Morphol 1975 Aug 01;341:93-112.

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The developmental capacity of nuclei transplanted from keratinized skin cells of adult frogs.

Gurdon JB , Laskey RA , Reeves OR .

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Nuclei from keratinized skin cells of adult Xenopus foot-webs have been transplanted to enucleated eggs of the same species. 2. The cells used to provide donor nuclei were obtained as a monolayer outgrowth from cultured foot-web explants. When explants were cultured without plasma for 3 days, over 99-9% of the outgrowth cells contained keratin as revealed by the binding of monospecific fluorescent antibody prepared against purified Xenopus keratin. Nuclei were transplanted from cells which had been cultured for 31/2 days. 3. None of the first transfer embryos developed as far as tadpoles. Eleven clones of embryos were prepared from the nuclei of partial first-transfer blastulae by use of serial nuclear transplantation. Eight of these clones contained swimming tadpoles with functional muscle and nerve cells, and six clones contained tadpoles with beating hearts, well differentiated eyes, and other organs. 4. To prove that the nuclei of nuclear-transplant tadpoles were derived from the transplanted skin cell nuclei and not from a failure of ultraviolet light to inactivate the recipient egg nucleus, 1-nu skin cell nuclei were transplanted to eggs laid by 2-nu frogs. Several advanced tadpoles from six clones were analysed for nucleolar and chromosome number and found to be 1-nu diploids. 5. The six clones of advanced tadpoles which were proved to carry the donor nuclear marker represent six first-transfer nuclei in a total sample of 129 skin cell nuclei originally transplanted. The probability that all six nuclei were derived from the 0-1% of the donor cell population not proved to contain keratin is less than one in 10(10). 6. We conclude that cell specialization does not involve any loss, irreversible inactivation or permanent change in chromosomal genes required for development.

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	FIGURE 1 Differentiation of adult Xenopus skin cells, used as nuclear donors. A, C, E, Phasecontrast photographs (x 325) of living cells: A = skin; C = lung and E = cultured cell line. B, D, F, ultraviolet fluorescence photographs (x 375) of cells after binding of fluorescent antibody prepared against purified frog keratin. B = skin, D = lung and F = cultured cell line. Skin cultures A and B were monolayer outgrowths of \-nu cells from foot web epidermis cultured according to 'without plasma' conditions specified in Methods. These cultures were photographed and tested for antibody-binding 3 days after the explant was made. The original explant was removed and all cells shown are those which migrated out from the explant. Only cells in such outgrowth areas were used to provide nuclei for transplantation (see Methods, p. 4). In B, all cells bound the anti-keratin antibody. C, D, Cells grown out from 1-nu lung tissue, photographed live (C) and tested for antibody-binding 3 days after the explant was made (D). E, F, A cultured line of cells which originated from wild-type, 2-nu embryos.
	Fig. 2. Plan of serial nuclear transfer experiments, using nuclei from adult skin celJs. Reasons for the various steps are explained in the text (p. 99). The percentages of injected eggs which cleave in different ways are those typically obtained with adult skin cell nuclei. The actual results of the experiments reported here are shown in Tables 1 and 3.
	FIGURE 3 Histological structure of a tadpole obtained by serial nuclear transplantation from a skin cell grown from a foot web under the 'without plasma' conditions specified in the Methods section. (A) Whole tadpole showing eye, heart, intestine, myotomes and melanophores. x 22. (B) Enlarged view of myotomes showing regular arrangement, x 62. (C) Transverse section through head region of tadpole showing nerve cord (nc), notochord (noto), otocysts (oto), and myotomes (myo). x 88. (D) Pronephros, showing nuclei with single nucleoli. x 308. (E) Intestine, with epithelium, x 616. (F, G) Sections through eye showing three-layered retina (G), lens body (lens), lens epithelium (epithel), inner and outer (ipl and opl) plexiform layers of retinal ganglion axons (Fig. G), retina, visual layers, and pigmented tapetum. x 308.
	Fig. 4. Sections of a swimming tadpole similar to that shown in Fig. 3 A, which resulted from the serial transplantation of a nucleus from keratinized skin cells cultured without plasma. (A) Organized blocks of muscle in the myotomes, and typically vacuolated cells of the notochord. x 4480. (B) A longitudinal section through a body muscle showing the characteristic cytological zones, x 21600. (C) A transverse section through tail muscles showing thick myosin filaments, each surrounded by six thin actin filaments, x 43 200. (D) Two sections through the nerve chord showing the characteristic microtubules (about 25 nm in diameter) in each sectioned axon. x 72000.