Nuclear transplants and genomic equivalence - John Gurdon’s expts

Editing Nuclear transplants and genomic equivalence - John Gurdon’s expts

An overview of the article “Developmental Capacity of Nuclei Transplanted from Keratinized Skin Cells of Adult Frogs” by John Gurdon, Ronald Laskey, and O. Raymond Reeves, published in 1975 in the Journal of Embryology and Experimental Morphology , Vol 34 (1) pp 93-112. This article was of great historical significance in the field of embryology, as it provided evidence for the concept of genomic equivalence.

Introduction

Visual representation of the basic steps involved in nuclear transplantation. Source: http://cc.scu.edu.cn/G2S/Template/View.aspx?courseType=1&courseId=17&topMenuId=113307&menuType=1&action=view&type=&name=&linkpageID=113785

Genomic equivalence is an important concept in developmental biology. Genomic equivalence is the principle that all cells in the body share the same genetic information (or genome) encoded in their DNA. Thus DNA is not lost during cellular division and the subsequent process of differentiation through which cells acuire their final adult state. The validity of the theory of genomic equivalence has been substantiated by many experiments over the years (with few exceptional cell types that do not retain the entire complement of the genome) (1). Some of the first researchers to provide significant evidence supporting this were Gurdon, Laskey, and Reeves, in 1975, in an experiment utilizing skin cells taken from a frog (2).

At the time of this experiment, it was still unclear what mechanisms were at work when an organism developed and cells differentiated into different types, such as heart, lung, skin, or other organs with varied functions. One theory postulated that a portion of the total DNA was removed or somehow altered during differentiation to create the modified instructions for a given type of specialized cell.

Gurdon and his colleagues set out to determine whether or not a fully differentiated cell from an adult organism contained the chromosomal material necessary to produce a complete and viable embryo. Their groundbreaking experiment utilized a technique called nuclear transplantation. This process is performed by removing or otherwise destroying the nucleus of an egg cell, so that a nucleus from a differentiated adult cell may be injected into it. The newly created cell is then allowed to develop to determine if the genetic information provided by the adult nucleus is able to grow into a functional organism with all the adult cell types. The work built on similar experiments performed earlier but in these experiments Gurdon et al used keratinized skin cells as their starting material instead of intestinal cells or cells from other regions of the body that might potentially contain relatively undifferentiated nuclear material. This provided a level of certainty not previously obtained that the implanted nuclei contained material from differentiated cells.

Materials and Methods

To obtain the donor nuclei used in transplantation, Gurdon et al. obtained 2 mm x 2 mm explants of skin and cultured the cells using two different solutions. The first culture, referred to as ‘with plasma’, was made up of two-thirds diluted chicken plasma covered by a two-thirds diluted Leibovitz L-15 medium, and it was incubated at 25 °C. The second culture, referred to as ‘without plasma’, was made up of two-thirds diluted Dulbecco’s modified Eagle’s medium, and it was incubated at 22 °C. After three days, these cultured cells were then exposed to fluorescent antibodies, which had been produced from purified Xenopus keratin. The cells that were cultured without plasma were found to be bound by the antibodies 99.9% of the time indicating that the cells had become fully differentiated. When Gurdon took the nuclei from these cultured cells, he therefore had a 99.9% chance of taking a nucleus from a differentiated skin cell.

To ensure that nuclear transplantation truly took place, Gurdon et al used single nucleolar nuclei as donors and inserted them into egg cells whose nuclei contained two nucleoli. The nuclear transplantation was done by exposing the animal pole of egg cells to UV radiation in order to destroy the nuclei, and then removing the donor nuclei from the adult skin cells with microforge-sharpened pipettes. The adult nucleus was then injected into the enucleated egg cells. These modified eggs were then allowed to develop until the point that they were partially cleaved. Once eggs were partially cleaved, the nuclei were again extracted and now inserted into a different enucleated egg completing what is known as a ‘serial transplantation’. The nuclei of first transfer eggs that either had become fully cleaved or had failed to cleave were not used for the second transfer. The second transfer eggs were then allowed to develop to tadpoles and analyzed. The reason they did the serial transfer was because the extent of cellular reprogramming was lower if only one round of nuclear transplantation was carried out.

Experiment

In this experiment the amphibean Xenopus laevis, or African clawed frog, was used as the experimental organism. Keratinized skin cells of the Xenopus foot web were isolated and grown without plasma. The presence of keratin in these cells were confirmed by fluorescent antibody staining. Donor eggs, from the same species, were then enucleated and grown for three and a half days.

Nuclei from the keratinized skin cells were then extracted and inserted into the donor eggs. While none of the initial clones developed as far as tadpoles, additional nuclei were isolated from these blastulae and used for serial nuclear transplantation. These embryos derived from the serial transplantation were successful in forming into swimming tadpoles with beating hearts, well differentiated eyes and other organs.

To prove the clones developed from the implanted skin cell nuclei and not from a failure to enucleate donor eggs, the foot web cells were taken from organisms with a single nucleolus while the donor eggs came from organisms with two nucleolei. Several highly developed tadpoles were tested and found to have a single nucleolus proving the genetic information for development came from the implanted skin cell nuclei.

Significance/Purpose

When cell differentiation occurs during development, the nuclei of the differentiated adult cells still retain all of the DNA that was originally found in the fertilized zygotes. These experiments suggest that there is neither loss of DNA nor irreversible inactivation of genes during the specification process of an embryonic cell to a somatic skin cell. These experiments also prove that most of the machinery that is required to develop an organism is kept within the nucleus of the cell. However, there must be other factors at play during the development of an organism from a single nucleus rather than just the DNA present. While this experiment was carried out with different enucleated eggs from different individuals and they each had successful transplantations, not every egg developed successfully. Most importantly, it seems that the nuclei has a short window to transition between non-dividing to the initiation of chromosomal replication. This being said, this experiment and others like it can only ascertain the minimum ability for an enucleated egg to produce a functional organism when a nucleus is placed into it. However, differentiated cells of an organism still contain the totality of chromosomal material that was present when the zygote first formed (1).

These results have been monumental in the scientific community. The discovery that a cell can be reverted back to its embryonic, or pluripotent, state with the full potential to develop into an entire organism has been repeatedly utilized in genetic and medical research. Further research has tried to find ways to convert one cell back to its pluripotent with some success. This induced state of pluripotency has lead to the creation of induced pluripotent stem (iPS) cells, which could have a myriad of uses in the medical field (3). One application would be in tissue donation. Organ transplants are risky due to the high frequency of rejection, where the patient's body rejects the donated organ due to the "non-self" cells. If iPS cells could be generated from the patient and grown into the necessary tissue, such as a kidney or skin, there would be a much smaller chance that the tissue would be rejected. Since the tissue is the patient's own tissue, the organ would be a perfect match (4). Our understanding of genomic equivalence has opened many doors for genetic and medical research that has a wide array of clinical applications that before were only wishful thinking.

References

1. Gilbert, Scott F. (2010). Developmental Biology (9th ed.). Sunderland, MA: Sinauer Associates, Inc.

2. J. B. Gurdon, R. A. Laskey, and O. R. Reeves (1975, August). The developmental capacity of nuclei transplanted from keratinized skin cells of adult frogs. Journal of Embryology and Experimental Morphology, 34, 93-112.

3. "The 2012 Nobel Prize in Physiology or Medicine - Press Release". Nobelprize.org. Nobel Media AB 2013. Web. 3 Mar 2014. <http://www.nobelprize.org/nobel_prizes/medicine/laureates/2012/press.html>

4. What are the potential uses of human stem cells and the obstacles that must be overcome before these potential uses will be realized? . In Stem Cell Information [World Wide Web site]. Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services, 2009 [cited Monday, March 03, 2014] Available at <http://stemcells.nih.gov/info/basics/pages/basics6.aspx>

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