Improving CRISPR/Cas9 mutagenesis efficiency by delaying the early development of zebrafish embryos.

CRISPR/Cas9 driven mutagenesis in zygotes is a popular tool for introducing targeted mutations in model organisms. Compared to mouse, mutagenesis in zebrafish is relatively inefficient and results in somatic mosaicism most likely due to a short single-cell stage of about 40 min. Here we explored two options to improve CRISPR/Cas9 mutagenesis in zebrafish-extending the single-cell stage and defining conditions for carrying out mutagenesis in oocytes prior to in vitro fertilization. Previous work has shown that ovarian fluid from North American salmon species (coho and chinook salmon) prolong oocyte survival ex vivo so that they are viable for hours instead of dying within minutes if left untreated. We found that commonly farmed rainbow trout (Oncorhynchus mykiss) ovarian fluid (RTOF) has similar effect on zebrafish oocyte viability. In order to prolong single-cell stage, we incubated zebrafish zygotes in hydrogen sulfide (H2S) and RTOF but failed to see any effect. However, the reduction of temperature from standard 28 to 12 °C postponed the first cell division by about an hour. In addition, the reduction in temperature was associated with increased CRISPR/Cas9 mutagenesis rate. These results suggest that the easily applicable reduction in temperature facilitates CRISPR/Cas9 mutagenesis in zebrafish.

Levels of some biological risk indicators among elderly men in Finland.

A 25-year follow-up survey of the Finnish men examined in the Seven Countries Study and now 65-84 years old was carried out in the East and the South-West of Finland in 1984. The follow-up examinations were carried out as in the previous surveys. Systolic and diastolic blood pressures were now significantly lower in the East than in the South-West of Finland. Serum total cholesterol and HDL-cholesterol were on the same level in both areas. The East/South-West difference in serum cholesterol, observed in previous studies, had levelled off and that in the blood pressure level had even reversed among the study cohorts. The mean fasting serum glucose was higher in the East than in the South-West of Finland. The mean serum calcium level was the same in both areas.