A novel epigenetic mechanism in early embryogenesis

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Our parental proposal aims to investigate critical mechanisms in mammalian early embryogenesis, such as endogenous transposable elements (TEs) remnants of ancient transposons and DNA methylation. We use embryonic stem cell (ESC) culture, genetic- engineered mouse model and in vitro fertilization (IVF) mouse models to interrogate the transcription and epigenetic regulatory pathways in ESC or developing mouse fetus. The current proposal advocates the needs for purchasing a versatile real-time PCR machine to accurately quantify the gene expression, epigenetic factors and genotype in early embryonic and extraembryonic tissues (Aim 1 and 2) and run protein thermal shift assays for biochemistry experiments (Aim3). Nearly half of our samples are generated from limited amounts of materials, such as 100-200 cells from morula or blastocyst stage mouse fetus. Our targets are often challenging, such as TEs that are highly repetitive. These unique research needs demand a precise and reliable instrument which also has the capability to run the Taqman assays as an orthogonal approach to independently validate the results from SYBR green-based real-time PCR assays. Furthermore, our experiments often require different throughputs, which need to interchange between a 96-well and 384-well plates. For example, a routine examination of gene expression level in embryonic stem cells needs a routine SYBR-green based assay in 96-well plates, whereas an examination of TE expression levels in mouse blastocyst needs Taqman assays in 384-well plates. Moreover, a major direction of our parental proposal is to investigate the function of SATB1, a critical remodeling factors binding to DNA, as well as novel DNA methyltransferases (Aim3). Thus, we need to an instrument that can run protein thermal shift assays to check protein quality and their binding to DNA substrates as shown in our recently published studies. This proposal aims to acquire an accurate, versatile and fast Real-Time PCR machine that meets multiple unique research needs in the parental proposal outlined above. The outcome of the proposal will greatly facilitate our investigation in epigenetics and embryogenesis.