To study genetic factors and the molecular mechanism underlying the development rate of mouse embryos, two mouse subspecies were studied. I have collected mouse embryos from crosses between C57BL6/JxCAST/EIJ (B6xCast), CastxB6 (the reciprocal cross), and intercrosses within either strain to examine the effects of maternal and paternal genomes on the developmental rate. I used mouse embryos every 24 hours at gestational stages E11.5-E14.5. Staging by the Embryonic Mouse Ontogenetic Staging System (eMOSS) is used to estimate the actual developmental stage based on limb bud morphology and compared to the gestational stage based on the estimated time of conception by observation of a vaginal plug. I also used gDNA to determine the sex of each embryo to investigate whether sexes play a role in developmental rate. I observed an interesting pattern that the mouse subspecies and the source of each parental genome affect the developmental rate of embryos. I observed embryos with at least one Cast parent, have faster development than B6xB6 starting at gestational stage E13.5. The developmental rate is independent of the sex of embryos or the litter size. Single-cell transcriptomic analysis is ongoing to determine the genes and mechanisms behind the change in developmental rate of mouse embryos. It is important because the result will contribute to human development in early stages, and help to solve problems such as preterm or underdeveloped infants.

X inactivation is a mechanism of dosage compensation that equalizes gene expression between males (XY) and females (XX). It is mediated by the long non-coding RNA (lncRNA) Xist (X-inactive specific transcript). However, some genes escape X chromosome inactivation(XCI) in females, and thus potentially contribute to sex differences. Escape genes are often identified by allelic expression analysis based on RNA sequences and RNA FISH (fluorescent in situ hybridization) based on imaging. In addition, RNA FISH allows the examination of escape status and the location of the transcript in individual cells. Allelic expression analysis has shown that one X-linked gene Car5b escapes XCI in mouse cells. However, it is not clear whether it escapes XCI in every cell or a portion of the cells. The goal of this project is to use RNA FISH to detect and quantify the escape status of Car5b in wild-type cells and in cells carrying a deletion of an important DNA element required for Car5b escape. In order to achieve the goal, I first prepared the fluorescent probes using the cDNA plasmids of Car5b and Xist. Next, dual RNA FISH was performed for Car5b and Xist in mouse cells to visualize the location of Car5b RNA signals whereas Xist RNA signals serve as the marker of inactivated X chromosome. Analyzing the FISH results is ongoing and we expect to visualize Car5b escape in the wild-type cells but not in the mutated cells. This method can be applied to escape studies in other X-linked genes which could enhance our understanding of X-linked gene regulation and sex difference.