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An embryonic cloning study: the viability of twinned Lytechinus variegates embryos K.A. LaSalle and B.C. Rakowski Swarthmore College


In the experiment, early stage sea urchin (Lytechinus variegates) embryos were twinned multiple times to determine the extent of viability of the undifferentiated cells. Four-cell blastomeres were separated and observed to determine their viability. Three samples of urchin embryos that had been twinned once, twice and three times, respectively, were observed through early development. "Viability," for this experiment has been defined as development to the pluteus stage, approximately twenty-four hours after fertilization. It was hypothesized that as the number of twinning events increases, the extent of viability of the embryos would decrease, due to the mosaic nature of sea urchin development.


Sometimes called "embryo cloning," embryo twinning is the process of separating blastomeres at the two- or four- celled stage in order to obtain genetically identical embryos. This process occurs naturally in utero in cases where identical twins are born. However, recently it has been attempted in clinical settings to observe the viability of twinned embryos. Several different methods have been attempted, including passing the embryos through a fine wire mesh, using fine wire or hair to squeeze the cells apart, or agitating the embryos until the blastomeres separated (Pence, 1998). Theoretically, the embryos of some species could be twinned indefinitely. However, in reality, extended twinning has been successful a very few times, if at all (Pence, 1998). Once cells have begun to differentiate, they are no longer totipotent due to the activation of localized transcription factors. Events such as the mid blastula transition (as in the case of sea urchins) or early stages of cellular differentiation (as in the two celled human embryo) impede the ability of twinned cells to develop into complete organisms (Gilbert, 2003).

In this experiment, sea urchins, (Lytechinus variegates) are used to examine the viability of twinned embryos in organisms that do not exhibit regulative development. Sea urchins were selected as the organism of choice for several reasons: large numbers of gametes are produced and easily harvested, fertilization and development occur externally, eggs are easily visible under a microscope, development occurs rapidly, and there is not a large yolk pole that could cause complication in the twinning process (Gilbert, 2003). Although similar experiments have been performed using mammalian embryos, the results of this experiment cannot be extrapolated to mammals. Sea urchin development, unlike regulative mammalian development, is mosaic. In mosaic development, cytoplasmic determinants are proportioned unequally among blastomeres after the first few cell divisions. Complete development of a separated blastomere cannot occur if the blastomere does not contain all of the necessary determinants. Evidence of this can be seen in pluteus larva developed from separated blastomeres: regions of the pluteus are truncated or incomplete. In contrast, mammalian embryos remain totipotent longer, even though embryonic transcription factors are turned on earlier in development (Gilbert, 2003).

In this experiment, several sea urchin embryos were twinned using a fine nylon mesh and the viability of the embryos created through the twinning process were be obsessed. The gametes were harvested using KCL injections in adult sea urchin embryos. The second cell division occurs 1.5-2.5 hours after fertilization, at which point the embryos were twinned. The twinning process was repeated three times as the embryos continued to develop. It was expected that viable pluteus could be obtained after the first and second twinning events, but that as the number of twinning events increases, the viability of the embryos decrease. For the purposes of this qualitative study, "viability" is defined as development to the pluteus larva stage, which should occur within twenty four hours of fertilization.

© 2003 Cebra-Thomas
Last Modified: 10 May, 2004

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