Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.

Sepsis is a clinical syndrome defined as a systemic inflammatory response to infection. Eritoran is a synthetic lipid A derivative that competes with lipopolysaccharide in binding to the identical site of myeloid differentiation-2/toll-like receptor 4 complex. Eritoran is effective in decreasing the septic mortality of Gram-negative bacteria-infected animals. Eritoran has been highlighted as a candidate drug for treatment of endotoxemia in phase I clinical studies with healthy human volunteers. A phase II trial of eritoran has been conducted in patients with severe sepsis. Intravenous infusion of eritoran reduced the mortality rate, as compared with the placebo group, in sepsis patients at a high risk of mortality according to acute physiology and chronic health evaluation-II scores. A phase III study of eritoran was completed in 2011. The results appear to be disappointing as no statistically significant difference in all-cause mortality was observed between the eritoran treatment group and the placebo group on day 28 in sepsis patients with a high risk of death. In this review, we focus on the rationale for the use of eritoran in treatment of sepsis as well as its clinical applications.