Effects of RNAi-mediated knockdown of Xist on the developmental efficiency of cloned male porcine embryos

Abstract

Xist is an X-linked gene responsible for cis induction of X chromosome inactivation. Studies have indicated that Xist is abnormally activated in the active X chromosome in cloned mouse embryos due to loss of the maternal Xist-repressing imprint following enucleation during somatic cell nuclear transfer (SCNT). Inhibition of Xist expression by injecting small interfering RNA (siRNA) has been shown to enhance the in vivo developmental efficiency of cloned male mouse embryos by more than 10-fold. The purpose of this study was to investigate whether a similar procedure can be applied to improve the cloning efficiency in pigs. We first found that Xist mRNA levels at the morula stage were aberrantly higher in pig SCNT embryos than in in vivo fertilization-derived pig embryos. Injection of a preselected effective anti-Xist siRNA into 1-cell-stage male pig SCNT embryos resulted in significant inhibition of Xist expression through the 16-cell stage. This siRNA-mediated inhibition of Xist significantly increased the total cell number per cloned blastocyst and significantly improved the birth rate of cloned healthy piglets. The present study contributes useful information on the action of Xist in the development of pig SCNT embryos and proposes a new method for enhancing the efficiency of pig cloning.

Fig. 1.

Comparison of Xist mRNA levels at different pre-implantation stages in pig SCNT and IVV-derived embryos. Xist mRNA levels in the IVV group were measured from 10–15 embryos (in theory, containing a mixture of male and female embryos at a ratio of 1:1), which were collected at the 8-cell (72 h post fertilization), 16-cell (96 h post fertilization), and morula (120 h post fertilization) stages. Xist mRNA levels in the SCNT group were measured from a mixture of 10–18 embryos (male:female = 1:1), which were collected at the 8-cell (72 h post activation), 16-cell (96 h post activation), and morula (120 h post activation) stages. At each stage, the Xist mRNA level of the SCNT group was normalized to that of the IVV group, which was defined as 1. ** The mean values, calculated from 3 replicates, are significantly different between the 2 groups (P < 0.01).

Fig. 2.

Screening for the most effective anti-Xist siRNA. A: Structural illustration of the porcine Xist gene showing the target site of the 3 designed anti-Xist siRNAs. B: Structural illustration of the plasmid expressing anti-Xist siRNA target sequences fused with the EGFP gene. The anti-Xist siRNA target site is about 400 bp, composed of partial sequences from exons 1, 5, and 6 of the pig Xist, and covers the target sequences of 3 designed anti-Xist siRNAs. C: Fluorescence microscopy analysis of EGFP expression, 48 h after co-transfection with anti-Xist siRNA target-EGFP fusion gene expression plasmid and siRNAs into HEK 293 cells. D: Flow cytometry of relative EGFP+ cell percentage, 48 h after co-transfection with the anti-Xist siRNA target-EGFP fusion gene expression plasmid and siRNAs into HEK 293 cells. E: Inhibition of Xist expression in female pig fibroblasts by 3 different siRNAs. ** The mean values, calculated from 3 replicates, are significantly different between the 2 groups (P < 0.01).

Fig. 3.

Inhibition of Xist expression at different pre-implantation stages of male pig SCNT embryos injected with anti-Xist siRNA1. Xist mRNA levels in each group were measured from a mixture of 10–20 embryos collected at the 4-cell (48 h post activation), 16-cell (96 h post activation), and morula (120 h post activation) stages. At each stage, Xist mRNA levels in the 5 µM and 50 µM siRNA1-injected groups were normalized to that in the non-injected (control) group, which was defined as 1. ** The mean values, calculated from 3 replicates, are significantly different between the 2 groups (P < 0.01).

Fig. 4.

Inhibition of Xist expression at different pre-implantation stages of male pig SCNT embryos injected with CM siRNA1. Xist mRNA levels in each group were measured from a mixture of 10–20 embryos collected at the 4-cell (48 h post activation), 16-cell (96 h post activation), and morula (120 h post activation) stages. At each stage, Xist mRNA levels in the CM siRNA1-injected group were normalized to that in the non-injected (control) group, which was defined as 1. * The mean values, calculated from 3 replicates, are significantly different between the 2 groups (P < 0.05); ** The mean values, calculated from 3 replicates, are significantly different between the 2 groups (P < 0.01).