Nuclear transplantation

Nuclear transplantation technique. The nucleus of an unfertilized egg is mechanically removed with a micropipette (.left), and a nucleus from a blastula cell is removed and microinjected into the enucleated egg. 

Gurdon, J. (1991). Nuclear transplantation in Xenopus. Methods in Cell Biol. 36,299-309. 

Na+/Ca2+ exchange, 260 Na+/K+ ATPase, 259-262 Neutral networks, 188-191 Nitrogenase, 212, 213 assay system, 212, 213 Nuclear transplantation, 268... 

Hakelien AM, et al. Reprogramming fibroblasts to express T-cell functions using ceU extracts. Nat. Biotechnol. 2002 20 460 66. Lanza RP, et al. Generation of histocompatible tissues using nuclear transplantation. Nat. Biotechnol. 2002 20 689-696. 

Wakamatsu, Y., B.S. Ju, I. Pristyaznhyuk, K. Niwa, T. Ladygina, M. Kinoshita, K. Araki and K. Ozato. Fertile and diploid nuclear transplants derived from embryonic cells of a small laboratory fish, medaka (Oryzias latipes). Proc. Natl Acad. Sci. USA 98 1071-1076, 2001. 

More recent studies using far more sensitive assays demonstrated that the onecell embryo is both transcriptionally competent and transcriptionally active. A series of nuclear transplantation experiments revealed that the one-cell embryo has a... 

Figure 1. Demonstration of transcriptional competence of one-cell embryos by nuclear transplantation. Donor two-cell nuclei from a-amanitin-treated embryos are transferred to enucleated one-cell embryos in which both the male and female pronuclei have been removed. Following transplantation, the cells are analyzed for TRC synthesis was shown to occur at a time that corresponds to C2 of the one-cell stage. Cytochalasin D, which has no effect on ZGA, is used to generate these donor cells since it is easier to remove nuclei from such treated embryos.

Figure 4. Demonstration of the development of a transcriptionally repressive state by nuclear transplantation. In the first experimental protocol, the injected nucleus was transplanted to an S-phase-arrested, one-cell embryo that was then analyzed at a time that corresponded to the mid-late two-cell stage. In the second protocol, the injected nucleus was transplanted to an S-phase-arrested, two-cell embryo that was analyzed at a time that corresponded to the four-cell stage, whereas in the third protocol the injected nucleus was transplanted to a two-cell blastomere that divided. In the last protocol, the injected two-cell blastomere in G2 was transplanted to an S-phase-arrested, one-cell embryo that was then at a time that corresponded to the mid-late two-cell stage. The data are expressed relative to the amount of activity observed for the tk promoter-containing reporter gene in the S-phase-arrested one-cell embryos, and were taken from Henery et al., 1995.

Nuclear transplantation experiments also indicated that the repression of expression from a plasmid-bome reporter gene that was observed following injection of the plasmid into a two-cell blastomere nucleus was essentially irreversible (Henery et al., 1995)(Figure 4). Transfer of a two-cell blastomere nucleus that was injected in G2 with the enhancerless promoter to an enucleated S-phase-arrested, one-cell embryo resulted in only 5% the level of expression when compared to S-phase-arrested, one-cell embryos, and this expression was ineffectively stimulated by the presence of an enhancer. This irreversible repression seemingly contradicted a previous report that transplantation of a nucleus from an eight-cell embryo or nuclei from differentiated cells to an enucleated one-cell embryo resulted in the re-expression of theTRC when examined at the two-cell stage (Latham et al., 1991) the TRC is apparently irreversibly repressed since its expression is not observed in... 

Hochedlinger K, Jaenisch R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. N. Engl. J. Med. 2003 349(3) 275-286. 

In nuclear transplantation it is the DNA that is hoped to be the same in every individual in a clone. However, the ovum used for the transplantation contains mitochondria. Some mitochondria may also accompany the nucleus during the transfer. If the donors of the ovum and of the nucleus are different individuals the offspring will be mitochondrial hybrids. In addition, there are questions about the methylation state of DNA in the donated nucleus and about the age and health of the donated mitochondria. That these questions are significant is emphasized by a bit of 3000-year-old knowledge from mule breeders a mare crossed with a donkey yields a mule but a stallion crossed with a donkey yields a hinny, which has shorter ears, a thicker mane and tail, and stronger legs than does a mule. There are worries because Dolly and many other animals produced by nuclear transplantation have not been completely healthy. ° °... 

Rideout W M, 3rd, Hochedlinger K, Kyba M, et al. (2002). Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell. 109 17-27. 

What governs the start of S phase There are several observations that indicate the onset of DNA synthesis in the cell cycle requires the prior synthesis of inducer molecules. There are the nuclear transplantation experiments (Graham et al., 1966 Guttes and Guttes, 1968 Ord, 1969) and cell fusion experiments (Rao and Johnson, 1970) that indicate these inducer molecules are present in the cytoplasm of cells synthesizing DNA but absent in cells not synthesizing DNA. Other observations concern the phenomenon of quantitative control. Only one copy of the DNA is made in each cell cycle, and the absence of reinitiation of DNA synthesis in G2 phase is best explained by the absence of these hypothetical inducers. 

Prescott and Goldstein (1969) discovered in Amoeba proteus a protein fraction which moves from nucleus to cytoplasm and vice versa as determined from nuclear transplantation experiments. It may be that these migrating proteins correspond to IFF and are involved in mRNA transfer. 

Gurdon, J. (1977) Methods for nuclear transplantation in amphibia. Meth. Cell Biol. 16, 125-139. 

Fig. 1. The transgenesis procedure includes (A) preparation of egg extracts, (B) sperm nuclei preparation, and (C) nuclear transplantation. The egg extracts and sperm nuclei can he stored at -80°C. A Calcium is added to allow the crude egg extract (which are held in meiotic arrest) to progress to interphase, and a high-speed centrifugation is performed to obtain the cytosolic fraction. B Testes are macerated and filtered, then the sperm suspension is treated with lysolecithin to disrupt the plasma membrane of the cells.

Restriction enzyme Dilute in water before adding to the transplantation reaction. Although any enzyme can be used, do not use an enzyme that digests within regulatory or protein coding regions of the construct. We usuaUy use enzymes, Notl, Sail, or Sfil purchased from Roche. Some cahbration may be required to determine the optimal amount of enzyme to add to each reaction, as too much enzyme may adversely affect the development of embryos derived from nuclear transplantations. 

Fig. 3. We use an oil-filled injection system for the nuclear transplantations. The syringe and tubing are filled with mineral oil and the infusion pump depresses the syringe plungw, resulting in a constant, desirable flow rate.

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