Single-cell analysis of nonhuman primate preimplantation development in comparison to humans and mice.

BACKGROUND: Genetic programs underlying preimplantation development and early lineage segregation are highly conserved across mammals. It has been suggested that nonhuman primates would be better model organisms for human embryogenesis, but a limited number of studies have investigated the monkey preimplantation development. In this study, we collect single cells from cynomolgus monkey preimplantation embryos for transcriptome profiling and compare with single-cell RNA-seq data derived from human and mouse embryos. RESULTS: By weighted gene-coexpression network analysis, we found that cynomolgus gene networks have greater conservation with human embryos including a greater number of conserved hub genes than that of mouse embryos. Consistently, we found that early ICM/TE lineage-segregating genes in monkeys exhibit greater similarity with human when compared to mouse, so are the genes in signaling pathways such as LRP1 and TCF7 involving in WNT pathway. Last, we tested the role of one conserved pre-EGA hub gene, SIN3A, using a morpholino knockdown of maternal RNA transcripts in monkey embryos followed by single-cell RNA-seq. We found that SIN3A knockdown disrupts the gene-silencing program during the embryonic genome activation transition and results in developmental delay of cynomolgus embryos. CONCLUSION: Taken together, our study provided new insight into evolutionarily conserved and divergent transcriptome dynamics during mammalian preimplantation development.

Single-cell RNA sequencing reveals distinct gene expression patterns in glucose metabolism of human preimplantation embryos.

Precise regulation of glucose metabolism-related genes is essential for early embryonic development. Although previous research has yielded detailed information on the biochemical processes, little is yet known of the dynamic gene expression profiles in glucose metabolism of preimplantation embryos at a single-cell resolution. In the present study, we performed integrated analysis of single-cell RNA sequencing (scRNA-seq) data of human preimplantation embryos that had been cultured in sequential medium. Different cells in the same embryo have similar gene expression patterns in glucose metabolism. During the switch from the cleavage to morula stage, the expression of glycolysis-related genes, such as glucose transporter genes (solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1) and solute carrier family 2 (facilitated glucose transporter), member 3 (SLC2A3) and genes encoding hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase, is increased. The genes involved in the pentose phosphate pathway are highly expressed at the cleavage stage, generating the reducing power to balance oxidative stress derived from biosynthesis. Expression of the genes involved in the biosynthesis of glycerophospholipids is increased after the morula stage. Nevertheless, the expression of tricarboxylic acid-related genes remains relatively unchanged during the preimplantation stages. In conclusion, we discovered that the gene expression profiles are dynamic according to glucose utilisation in the embryos at different stages, which contributes to our understanding of regulatory mechanisms of glucose metabolism-related genes in human preimplantation embryos.

[Silica nanoparticles modified as carriers for gene transfection].

The silica nanoparticles are modified by sodium iodine or sodium chloride with different concentration. Their ability to bind and protect plasmid DNA was demonstrated by agarose gel electrophoresis. The EGFP-N1 plasmid was transfected into HT1080 by combing silica nanoparticle. Eletromicroscope examine revealed that nanoparticle-DNA could enter into cells, these silica nanoparticles protect DNA against nuclease digestion and the GFP has an effective expression. In conclusion, these nanoparticles might be used as DNA carriers for gene transfection.

[Separation of adhering cell colonies with a direct digestion method].

OBJECTIVE: To establish a highly efficient method for the separation of adhering cell colonies. METHODS: Cell suspension was diluted gradiently to four different densities and then seeded on a cell culture plate covered with parted fibrinous membranes. After colonies appeared, they were digested directly with 0.25% trypsin and picked out with a pipette tip. RESULTS: At the density of 5-10.cm-2, colonies could be separated most efficiently in both continuous and normal diploid cell line colonies. The cell number could reach 10(6) after one month of culture for a continuous cell line colony and one and a half month of culture for a normal diploid cell line colony. CONCLUSION: With the direct digestion method, single cell colonies can be effectively separated from the plate covered with parted fibrinous membranes, when the cell was initially seeded at the density of 5-10.cm2.

[Apolipoprotein E gene expression in peripheral blood monocyte in children with obesity].

OBJECTIVE: Coronary heart disease (CHD) is one of the most common causes of death in the world. Some studies suggested that CHD begins in childhood. Obesity and dyslipidemia are important risk factors of coronary heart disease. Apolipoprotein (apo)E gene associated with dyslipidemia and coronary heart disease. The present study was designed to investigate the expression status of apoE gene in peripheral blood monocyte and association of apoE gene expression with lipids in children with obesity. METHODS: Among 32 children with obesity and 32 healthy children without obesity or overweight, ApoE gene expressions were determined by competitive reverse transcription-polymerase chain reaction in peripheral blood monocyte. The concentrations of plasma triglyceride, total cholesterol, low density lipoprotein-cholesterol, high density lipoprotein-cholesterol, lipoprotein(a), apoA I, apoB(100) and apoE were measured. RESULTS: Expression of apoE gene was detected in peripheral blood monocyte. Expression of apoE gene was significantly reduced in children with obesity as compared with control group (0.29 +/- 0.14 moles/mole GAPDH mRNA vs. 0.36 +/- 0.10 moles/mole GAPDH mRNA, t = 2.15, P < 0.05). The more severe was the degree of obesity, the more significantly reduced the expression of apoE gene; the degree of obesity was negatively correlated with the levels of expression of apoE gene (correlation coefficient = -0.40, P < 0.05). Compared with control group, the levels of triglyceride, total cholesterol, low density lipoprotein-cholesterol, and apoB(100) were higher, and those of high density lipoprotein-cholesterol, apoA I and apoE were lower in children with obesity [(1.68 +/- 0.50) mmol/L vs. (0.99 +/- 0.54) mmol/L, (4.47 +/- 0.91) mmol/L vs. (3.33 +/- 0.90) mmol/L, (2.23 +/- 0.71) mmol/L vs. (1.13 +/- 0.96) mmol/L, (94.48 +/- 9.97) mg/dl vs. (83.81 +/- 15.64) mg/dl, (1.47 +/- 0.39) mmol/L vs. (1.73 +/- 0.36) mmol/L, (112.71 +/- 27.86) mg/dl vs. (134.80 +/- 45.36) mg/dl, (24.50 +/- 10.92) mg/L vs.(35.07 +/- 9.79) mg/L, respectively, P < 0.05]. ApoE gene expression was associated with plasma lipids metabolism in children with obesity. The quantity of apoE gene expression was inversely associated with low density lipoprotein-cholesterol, positively correlated with apoE (correlation coefficient = -0.33, 0.35, respectively, P < 0.05). The quantity of apoE gene expression was not associated with total cholesterol, triglyceride, high density lipoprotein-cholesterol, lipoprotein(a), apoA I, and apoB(100) (correlation coefficient = -0.19, -0.11, 0.16, 0.09, 0.18, 0.22, P > 0.05). CONCLUSION: Expression of apoE gene was significantly reduced in peripheral blood monocyte in children with obesity. The quantity of apoE gene expression was associated with degree of obesity and abnormality of blood lipids.