Methods of karyoplast cell cycle synchronization for increasing the efficiency of somatic cloning of farm animals

Abstract

Somatic cloning is a method of obtaining genetically identical offspring, which for some reason have extremely low efficiency. Methods to increase the effectiveness of this procedure aimed at optimizing each of its stages, one of which is karyoplast preparation. In most experiments, to obtain cloned offspring, oocytes are used as recipient cells at the metaphase of the second meiotic division without prior activation, which determines the choice of somatic cells as karyoplasts at the G0/G1 stage, the most optimal for subsequent nuclear reprogramming by oocyte cytoplasmic factors. Serum starvation and/or contact inhibition are the most commonly used methods for arresting cells in this phase, which allows the arrest of up to 90% of cells at the G0/G1 stage. Despite the effectiveness of these methods, they have some significant limitations; therefore, the addition of components to the culture medium of somatic cells that prevent the progression of cells through the cell cycle stages is becoming widespread. Some chemical inhibitors have a protective effect on somatic cells, preventing the induction of apoptotic changes. Although the efficacy of butyrolactone I, mimosine, and aphidicolin application is controversial, several studies have attested the possibility of using these drugs to synchronize the karyoplasts. Thus, several methods can be employed for effective synchronization of the cell cycle of karyoplasts. When choosing the optimal method, the type of cells, species of animals from which they were obtained, and permissible duration of cultivation under given conditions must be considered to minimize the negative effect of conditions on karyoplast viability.

Keywords

SCNT / somatic cloning / cell cycle synchronization / roscovitine / rapamycin / serum starvation / contact inhibition

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