Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis

Abstract

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a 2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a 4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a 4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a 4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study.

Mouse Parthenogenetic Embryonic Stem Cells with Biparental-Like Expression of Imprinted Genes Generate Cortical-Like Neurons That Integrate into the Injured Adult Cerebral Cortex

“…For instance, a shRNA against H19 reduces its expression level while promoting cardiomyogenesis in Pg‐ESCs . Embryo aggregation also greatly improves the fidelity of imprinted gene expression of Pg‐ESCs .…”

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.…”

“…The plasmids used were as followed: psp73-KDM6A and GFP-C1-KDM6A were cloned from the cDNA of mouse ES cell line that established in our laboratory (Bai et al ., 2016). The antibodies covered were as followed: monoclonal anti-B23 antibody (Sigma, FC82291), monoclonal anti-UBF antibody (Sigma, WH00007343M1), polyclonal rabbit anti-H3K27me3 (Millipore, 07-449), monoclonal rabbit anti-PARP1 (Abcam, ab32138), monoclonal mouse anti-GAPDH (CW0266A, CWBiotech).…”

Long Non-coding RNA H19 Suppression Protects the Endothelium Against Hyperglycemic-Induced Inflammation via Inhibiting Expression of miR-29b Target Gene Vascular Endothelial Growth Factor a Through Activation of the Protein Kinase B/Endothelial Nitric Oxide Synthase Pathway

“…One of the first identified lncRNAs, H19, plays a wide range of roles in vivo . H19 can function as both a tumor suppressor or oncogene and also as a regulator of growth and development in multiple mammalian embryo tissues (Bai et al, 2016; Yoshimura et al, 2018). A previous study revealed that H19 is up-regulated in AS and is related to AS progression (Pan, 2017), but the underlying regulation mechanism has not yet been determined.…”