Effect of Additional Cytoplasm of Cloned Embryo on In Vitro Developmental Competence and Reprogramming Efficiency in Mice

Abstract

Somatic cell nuclear transfer (SCNT) is an important technique for biological science research. Cytoplasm injection cloning technology (CICT) was developed to improve the reprogramming efficiency as well as to overcome the limitations of SCNT. CICT uses an additional cytoplasm fused with an enucleated oocyte to restore the cytoplasmic volume of the cloned embryo, and this method could improve the reprogramming efficiency of the cloned embryo. In this study, we show that CICT can be adapted to mouse species to overcome the inefficiency of the SCNT method. In this study, results indicate that the two-cell embryo and blastocyst rates of cloned embryos with the use of the CICT method were significantly higher (p < 0.05) than that of the SCNT method (96.6%-1.1% vs. 86.7%-6.0%, 29.5%-2.6% vs. 22.1%-3.0%, respectively). Furthermore, the apoptotic cell number per blastocyst was significantly lower in the CICT group than that in the SCNT group (1.7-0.2 vs. 2.9-0.3, p < 0.05). Moreover, the acH3K9/K14 expression level in the CICT group was greater than that of the SCNT group (p < 0.05), and the relative acH3K56 level in the CICT group was significantly (p < 0.05) higher than that in the SCNT group. These results indicate that CICT helps improve the in vitro developmental competence and quality of cloned embryos.

Strategies to Improve the Efficiency of Somatic Cell Nuclear Transfer

“…Similarly, in cloned mice, cytoplasmic supplementation improved the in vitro development and quality of cloned mouse embryos. Additionally, CICT-derived mouse embryos had a lower number of apoptotic cells and higher acetylation levels when compared with those of SCNT-derived embryos [77]. Notably, CICT significantly increased the birth rate of cloned cat embryos when compared to that of SCNT (4.8% and 0.7%, respectively) [78].…”

Omne vivum ex ovo: the oocyte reprogramming and remodeling activities

“…Nevertheless, the question of the reprogramming factor-to-DNA ratio seems to be rather complicated and conflicting results can be found in the literature: on one hand, some authors reported that increasing the amount of the reprogramming factors present by MII cytoplast fusion is not beneficial in terms of the SCNT efficiency outcomes (Sayaka et al 2008). On the other hand, others claimed that introducing extra cytoplasm to the cytoplasts leads to better developmental rates to the blastocyst and increased H3K9/K14 acetylation (Song et al 2020). At the same time, the hand-made cloning, during which the egg is bisected into two halves and the half lacking the spindle is used as recipient cytoplast, has been used with success in large animals; for review, see Verma et al 2015, Saini et al 2018 Although the reduced size cytoplasts are not typically used in the laboratory mouse, the hand-made cloning raises the question of whether the full content of cytoplast reprogramming and remodeling factors is indeed needed to achieve the SCNT success and whether the cytoplast impacts the transferred nucleus only in a positive manner.…”

Development of somatic cell nuclear transfer biotechnology for cloning of animals: a mini-review

“…The possibility to improve the quality of cloned mice with the SCNT technique has been developed and is very useful for biomedical research purposes as an experimental animal (Ogura, 2020). Song et al (2020) cloned mouse embryos by reprogramming the cloned embryos' cytoplasmic volume. Cytoplasm injection (CICT; cytoplasm injection cloning technology) was developed to increase reprogramming of the SCNT.…”