Nuclear Transplantation

An important problem in embryology is whether the differentiation of cells depends upon a stable restriction of the genetic information contained in their nuclei. The technique of nuclear transplantation has shown to what extent the nuclei of differentiating cells can promote the formation of different cell types (e.g. King & Briggs, 1956; Gurdon, 1960c). Yet no experiments have so far been published on the transplantation of nuclei from fully differentiated normal cells. This is partly because it is difficult to obtain meaningful results from such experiments. The small amount of cytoplasm in differentiated cells renders their nuclei susceptible to damage through exposure to the saline medium, and this makes it difficult to assess the significance of the abnormalities resulting from their transplantation. It is, however, very desirable to know the developmental capacity of such nuclei, since any nuclear changes which are necessarily involved in cellular differentiation must have already taken place in cells of this kind.

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Efficient reactivation of Xenopus erythrocyte nuclei in Xenopus egg extracts

Journal of Cell Science ◽

10.1242/jcs.108.6.2187 ◽

1995 ◽

Vol 108 (6) ◽

pp. 2187-2196 ◽

Cited By ~ 3

Author(s):

L.J. Wangh ◽

D. DeGrace ◽

J.A. Sanchez ◽

A. Gold ◽

Y. Yeghiazarians ◽

...

Keyword(s):

Cell Cycle ◽

Somatic Cell ◽

Nuclear Transplantation ◽

Optimal Time ◽

Genome Replication ◽

Cell Nuclei ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Rapid genome replication is one of the hallmarks of the frog embryonic cell cycle. We report here that complete reactivation of quiescent somatic cell nuclei in Xenopus egg extracts depends on prior restructuring of the nuclear substrate and prior preparation of cytoplasmic extract with the highest capacity to initiate and sustain DNA synthesis. Nuclei from mature erythrocytes swell, replicate their DNA efficiently, and enter mitosis in frozen/thawed extracts prepared from activated Xenopus eggs, provided the nuclei are first treated with trypsin, heparin, and an extract prepared from unactivated, meiotically arrested, eggs. Optimal replicating extracts are prepared from large batches of unfertilized eggs that are synchronously activated into the cell cycle for 28 minutes (at 20 degrees C). Because the Xenopus cell cycle progresses so rapidly, extracts prepared just a few minutes before or after this time have substantially lower DNA synthetic capacities. At the optimal time and temperature, eggs have just reached the G1/S boundary of the first cell cycle. This fact was revealed by injecting and replicating an SV40 plasmid in intact unfertilized eggs as described previously. We estimate that under optimal conditions approximately 6.14 × 10(9) base pairs of DNA/per nucleus are synthesized in 30–40 minutes, a rate that rivals that observed in the zygotic nucleus. The findings reported here are one step in our long term effort to develop a new in vitro/in vivo approach to nuclear transplantation. Nuclear transplantation in amphibian embryos has been used to establish that the genomes of many types of differentiated somatic cells are pluripotent. But very few such nuclei have ever developed into advanced tadpoles or adult frogs, probably because somatic nuclei injected directly into activated eggs fail to reactivate quickly enough to avoid being damaged during first mitosis. We have already shown that unfertilized eggs can be injected prior to activation of the first cell cycle. Future experiments will reveal whether in vitro reactivated somatic cell nuclei transplanted into such eggs reliably reach advanced stages of development.

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Analysis of function of the pair-rule genes hairy, even-skipped and fushi tarazu in mosaic Drosophila embryos

Development ◽

10.1242/dev.107.4.847 ◽

1989 ◽

Vol 107 (4) ◽

pp. 847-853 ◽

Cited By ~ 1

Author(s):

P.A. Lawrence ◽

P. Johnston

Keyword(s):

Genetic Interactions ◽

Nuclear Transplantation ◽

Fushi Tarazu ◽

Segmentation Gene ◽

Hairy Cells ◽

Analysis Of Function ◽

Mutant Cells ◽

Pair Rule ◽

Drosophila Embryos

We report the first attempt of its kind to study genetic interactions using young Drosophila embryos that are mosaic for wildtype and mutant cells. Using nuclear transplantation we make mosaic embryos in which a patch of cells lacks a particular segmentation gene, A. With antibodies, we than look at the expression of another gene that is known to be downstream of gene A, with respect to the cells in the patch. We have examples of patches of hairy cells (where we monitor the effect on fushi tarazu (ftz) expression), even-skipped (monitoring ftz) and ftz (monitoring engrailed and Ultrabithorax). Our main finding is that the dependence of engrailed expression on the ftz gene is strictly cell-autonomous. This result goes some way towards explaining the dependence of Ultrabithorax expression on ftz, a dependence we show to be locally cell-autonomous within parts of parasegments 6 and 8 but non autonomous within parasegment 7.

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Gene reactivation in erythrocytes: nuclear transplantation in oocytes and eggs of Rana

Science ◽

10.1126/science.6600520 ◽

1983 ◽

Vol 219 (4586) ◽

pp. 862-864 ◽

Cited By ~ 51

Author(s):

M. DiBerardino ◽

N. Hoffner

Keyword(s):

Nuclear Transplantation ◽

Gene Reactivation

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Nuclear transplantation and the control of gene activity in animal development

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1970.0050 ◽

1970 ◽

Vol 176 (1044) ◽

pp. 303-314 ◽

Cited By ~ 81

Keyword(s):

Nuclear Transfer ◽

Somatic Cells ◽

Gene Activity ◽

Protein Fractions ◽

Nuclear Transplantation ◽

Animal Development ◽

Cytoplasmic Proteins ◽

Intracellular Movement ◽

Selective Accumulation ◽

Structure And Metabolism

The transplantation of nuclei from differentiated or determined somatic cells to enucleated frogs’ eggs consistently leads to a complete and clearly recognizable change of gene activity. Within 1 to 2 h of nuclear transfer, somatic nuclei have come to resemble in structure and metabolism the zygote nuclei of fertilized eggs. The change in gene activity therefore takes place very soon after nuclear transfer and results from an effect of egg cytoplasm. The induced change in gene activity is associated with a selective accumulation of cytoplasmic proteins in transplanted nuclei. Examples are given of various ways in which nuclear transplantation and microinjection can be used to elucidate the intracellular movement of proteins and the effect of known protein fractions on gene activity.

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Nuclear transplantation, the conservation of the genome, and prospects for cell replacement

FEBS Journal ◽

10.1111/febs.13988 ◽

2017 ◽

Vol 284 (2) ◽

pp. 211-217 ◽

Cited By ~ 4

Author(s):

J. B. Gurdon

Keyword(s):

Nuclear Transplantation ◽

Cell Replacement

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The pattern of appearance of enzymic activity during the development of the Golgi apparatus in amoebae

Journal of Cell Science ◽

10.1242/jcs.34.1.53 ◽

1978 ◽

Vol 34 (1) ◽

pp. 53-63

Author(s):

C.J. Flickinger

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Rough Endoplasmic Reticulum ◽

Enzymic Activity ◽

Nuclear Transplantation ◽

Similar Distribution ◽

Cytochemical Staining ◽

Light Reaction ◽

Golgi Bodies ◽

Golgi Membranes

The appearance of enzymic activity during the development of the Golgi apparatus was studied by cytochemical staining of renucleated amoebae. In cells enucleated for 4 days, there was a great decline in size and number of Golgi bodies, or dictyosomes. Subsequent renucleation by nuclear transplantation resulted in a regeneration of Golgi bodies. Samples of amoebae were fixed and incubated for cytochemical staining at intervals of 1, 6, or 24 h after renucleation. Enzymes selected for study were guanosine diphosphatase (GDPase), esterase, and thiamine pyrophosphatase (TPPase). All three were found in the Golgi apparatus of normal amoebae but they differed in their overall intracellular distribution. GDPase was normally present at the convex pole of the Golgi apparatus, in rough endoplasmic reticulum, and in the nuclear envelope. In amoebae renucleated for 1 h, light reaction product for GDPase was present throughout the small stacks of cisternae that represented the forming Golgi apparatus. By 6 h following the operation GDPase reaction product was concentrated at the convex pole of the Golgi apparatus. Esterase, which was distributed throughout the stacks of normal Golgi cisternae, displayed a similar distribution in the forming Golgi bodies as soon as they were visible. TPPase was normally present in the Golgi apparatus but was not found in the endoplasmic reticulum. In contrast to the other enzymes, TPPase reaction product was absent from the forming Golgi apparatus 1 and 6 h after renucleation, and did not appear in the Golgi apparatus until 24 h after operation. Thus, enzymes held in common between the rough endoplasmic reticulum and the Golgi apparatus were present in the forming Golgi apparatus as soon as it was detectable, but an enzyme cytochemically localized to the Golgi apparatus only appeared later in development of the organelle. It is suggested that Golgi membranes might be derived from the endoplasmic reticulum and thus immediately contain endoplasmic reticulum enzymes, while Golgi-specific enzymes are added later in development.

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