RNAi-mediated knockdown of Parp1 does not improve the development of female cloned mouse embryos

Abstract

Somatic cell nuclear transfer is an important technique for life science research, but its efficiency is still extremely low, and most genes that are important during early development, such as X chromosome-linked genes, are not appropriately expressed during this process. Poly (ADP-ribose) polymerase (PARP) is an enzyme that transfers ADP ribose clusters to target proteins. PARP family members such as PARP1 participate in cellular signalling pathways through poly (ADP-ribosylation) (PARylation), which ultimately promotes changes in chromatin structure, gene expression, and the localization and activity of proteins that mediate signalling responses. PARP1 is associated with X chromosome inactivation (Xi). Here, we showed that abnormal Xi occurs in somatic cell nuclear transfer (NT) blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. Parp1 expression was higher in female NT blastocysts than that in intracytoplasmic sperm injection (ICSI) embryos but not in male NT blastocysts. After knocking down Parp1 expression, both the pre-rRNA 47S and X-inactivation-specific transcript (Xist) levels increased. Moreover, the expression of genes on the inactivated X chromosome, such as Magea6 and Msn, were also increased in the NT embryos. However, the development of Parp1si NT embryos was impaired, although total RNA sequencing showed that overall gene expression between the Parp1si NT blastocysts and the control was similar. Our findings demonstrate that increases in the expression of several genes on the X chromosome and of rRNA primary products in NT blastocysts with disrupted Parp1 expression are insufficient to rescue the impaired development of female cloned mouse embryos and could even exacerbate the associated developmental deficiencies.

Kdm6a overexpression improves the development of cloned mouse embryos

“…Intracytoplasmic sperm injection (ICSI) was carried out by a piezo-driven unit using methods described elsewhere (Bai et al, 2017), except that our experiment was performed in HEPES-CZB containing 5 μg/ml cytochalasin B (Sigma, C6762) at room temperature. Only the sperm head was injected into the oocyte.…”

“…Embryos were collected at blastocyst stages. RNA extraction and qPCR evaluation were performed as previously described with some modifications (Bai et al, 2017). Total RNA was extracted from embryos using an RNeasy Mini Kit (Qiagen, 74104) according to the manufacturer's instructions.…”