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Kuznetsov A. V., Vainer V. I., Volkova Yu. M., Tsygankova V. M., Bochko D. N., Mukhanov V. S. Trichoplax sp. H2 cultivation and regeneration from body fragments and dissociated cell aggregates: Outlook for genetic modification. Marine Biological Journal, 2022, vol. 7, no. 3, pp. 60-79. URL: https://marine-biology.ru/mbj/article/view/353

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Abstract

Trichoplax sp. H2, a simple multicellular animal cultivated in the laboratory, was studied with the aim of its further genetic modification. The idea here is to introduce genetic information into a cell suspension after dissociation of the Trichoplax body into single cells, followed by their aggregation and regeneration of the resulting agglomerates into a viable animal. 1. We analyzed the dynamics of the Trichoplax growth in Petri dishes on Tetraselmis marina algal mats. Specimens were uniform on the exponential growth stage. 2. Trichoplaxes were cut radially in a post-traumatic regeneration research, and the regeneration of the obtained parts was investigated under a microscope. Growth and reproduction rate of animals on nutrient mats were determined that decreased as the animals had been cut. The missing part of the Trichoplax body was replaced by remodeling of remaining cells. 3. The animals after a vital staining were dissociated into single cells in a medium with no divalent cations. Pear-shaped or rounded cells were identified, as well as epithelial cells with flagella maintaining motion activity for more than 12 hours. 4. Trichoplax plates were disintegrated in the presence of 10 μM amlodipine to quantify a cell population using flow cytometry. As estimated, Trichoplax (0.5–1 mm in size) consists of approximately 10,000 cells. 5. Treatment of animals with 10 % BSA (Bovine Serum Albumin) during various exposure intervals suggests a hypothesis on the existence of totipotent cells at the periphery of the Trichoplax body, probably in the rim. 6. In the course of reparative regeneration experiments, we achieved Trichoplax dissociation into single cells with 0.1 % BSA treatment and the following recreation of the viable organisms by centrifugation of a cell suspension and subsequent dispersion of a large pellet into fragments up to 0.1 mm prior to plating multicellular aggregates on nutrient mats. 7. The development of the aggregates was accompanied by active motion of cells and epithelialization of the surface, which resulted in cell growth, formation of a plate, and further vegetative division of Trichoplax. As assumed, the artificial stage of a single cell in a line of asexual reproductions allows to introduce foreign genetic information into Trichoplax, for example, in order to study the signal processing, organization, and functioning of this multicellular organism. Transgenesis, which is based on the dissociation of an animal body into single cells, could be applied to other organisms with high regenerative potential.

Authors

A. V. Kuznetsov

leading researcher, D. Sc.

https://orcid.org/0000-0002-0015-7994

https://elibrary.ru/author_items.asp?id=1043295

V. I. Vainer
Yu. M. Volkova
V. M. Tsygankova
D. N. Bochko
V. S. Mukhanov

leading researcher, PhD

https://orcid.org/0000-0002-5324-045X

https://elibrary.ru/author_items.asp?id=986529

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Description: Trichoplax 30 minutes after bisection
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Description: Trichoplax 60 minutes after bisection
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