Retinoblastoma-binding protein 4 (RBBP4) is a subunit of chromatin remodeling factor 1 (CAF-1) and is essential for mammalian oocyte maturation, embryo survival, and embryo implantation. RBBP4 also localizes to the chromatin and is a ubiquitously expressed nuclear protein. Previous methods used to study this protein include short interfacing RNAs (siRNAs) and CRISPR/Cas9. These techniques have limitations such as determining an indirect depletion of proteins, may trigger compensatory mechanisms, and may not be useful in non-dividing primary cells. A new, acute, and rapid endogenous protein depletion technique called Trim-Away, can overcome these limitations. Trim-Away is also widely applicable since it can be used with many off-the-shelf reagents. Trim-Away utilizes the TRIM21-antibody interaction within the cytosol and the ubiquitin-proteasome pathway (UPP) to target and degrade a protein of interest. Studying RBBP4 using Trim-Away can offer insight into possible new functions of RBBP4 and its maternal effect, and increase the knowledge on a new, acute, and endogenous protein depletion technique. Here we report that, RBBP4 is required for proper blastocyst development and RBBP4 is more abundant in MII oocytes than GVBD oocytes. We also report that the loss of RBBP4 hinders RNA synthesis and causes cell death in later stages of embryo development. While our Trim-Away methodology can deplete RBBP4 as early as the 2-cell stage in embryos, our oocyte Trim-Away protocol needs to be optimized.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:masters_theses_2-2128 |
| Date | 01 July 2021 |
| Creators | Barletta, Holly L |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses |
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