Abstract
Abstract The success of cloning by somatic cell nuclear transfer depends on the efficiency of nuclear reprogramming, with the cycle stage of the donor cell playing a crucial role. Therefore, the aim was to evaluate three different approaches for cell cycle synchronization: (i) serum starvation (SS) for 1 to 4 days, (ii) contact inhibition (CI) for 1 to 3 days, and (iii) using cell cycle regulatory inhibitors (dimethyl sulfoxide, cycloheximide, cytochalasin B, or 6-dimethylaminopurine) for 1 and 2 days, in terms of their effects on synchronization in G0/G1 phases and viability of collared peccary skin fibroblasts. Flow cytometry analysis revealed that SS for 4 days (79.0% ± 1.6) and CI for 3 days (78.0% ± 1.4) increased the percentage of fibroblasts in G0/G1 compared to growing cells GC (68.1% ± 8.6). However, SS for 3 and 4 days reduced the viability evaluated by differential staining (81.4% ± 0.03 and 81.6% ± 0.06) compared to growing cells (GC, 95.9% ± 0.06). CI did not affect the viability at any of the analyzed time intervals. No cell cycle inhibitors promoted synchronization in G0/G1. These results indicate that CI for 3 days was the most efficient method for cell cycle synchronization in peccary fibroblasts.
Highlights
The collared peccaries are wild mammals endemic to the American continent, where they play a crucial ecological role in maintaining their habitats (Desbiez et al, 2012)
We have developed a suitable protocol for somatic cell synchronization, the last step in the preparation of karyoplasts, i.e., somatic nucleus donor cells
We identified that using contact inhibition (CI) for 3 days was more efficient for cell cycle synchronization when compared to serum starvation (SS) for 1 to 4 days and treatment with cell cycle inhibitors (DMSO, CHX, CB, or 6-DMAP) for 1 and 2 days
Summary
The collared peccaries are wild mammals endemic to the American continent, where they play a crucial ecological role in maintaining their habitats (Desbiez et al, 2012). Their population is not considered to be of concern internationally (Gongora et al, 2011), in some biomes, such as Caatinga and the Atlantic forest, these individuals’ populations are declining (Desbiez et al, 2012), requiring conservation strategies that ensure their maintenance in biodiversity This species’ high adaptation capacity and zootechnical performance in captivity (Briceño-Méndez et al, 2016) suggest that assisted reproductive technologies (ARTs) such as cloning by somatic cell nuclear transfer (SCNT) may be successfully used for generation and multiplication of genetically identical specimens of collared peccary (Pecari tajacu) as well as for research focused on elucidation of molecular and epigenetic mechanisms underlying embryonic development in this mammalian species. While SS and CI act on the checkpoints by depriving the cells of adequate environmental or nutritional conditions (Kues et al, 2000), cell cycle inhibitors regulate specific biosynthetic processes such as repression or induction of cyclins (dimethyl sulfoxide, DMSO), protein synthesis inhibition (cycloheximide, CHX), cytoskeleton inhibition (cytochalasin B, CB), and protein kinase inhibition (6-dimethylaminopurine, 6-DMAP) to cause prolongation of the G1 phase (Koo et al, 2009; Kretz et al, 2019)
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