Year: 2025
Type: preprint
Abstract: The purpose of this protocol is to genotype mouse blastocysts. Mouse blastocysts can be obtained by culturing fertilized eggs in vitro or by in vivo collection from female mice. Single mouse blastocysts contain about 64 cells, yielding a few picograms of genomic DNA. The DNA extraction protocol below is based on Sakurai et al. (2014). The result is a crude DNA extract that is then placed in a PCR reaction to detect genes or genetic changes of interest. Due to low DNA yields from single blastocysts, we use 29 nucleotide long PCR primers to increase specificity of PCR products so that only a single DNA band is amplified from genomic DNA as described (Stratman et al. 2003). We prefer to use a software driven approach to primer selection rather than guessing at primer sequences. For this purpose, we use the web-based Primer-BLAST tool at NCBI (Ye et al. 2012). Often, we find that GC rich DNA templates amplify with low efficiency. The addition of CES (Combinatorial PCR Enhancer Solution) to the PCR reaction mix can improve PCR efficiency of some difficult to amplify templates (Ralser et al. 2006). We use this method to determine if fertilized eggs treated with CRISPR Cas9 or other similar reagents carried designed genetic changes. In this way we can validate CRISPR reagents prior in our CRISPR pipeline before we use large numbers of mice to generate a novel mouse model for translational preclinical research.
Authors: Thom Saunders, Sivakumar Jeyarajan, Jennifer Leo, Eden A. Dulka, Honglai Zhang, Zachary Freeman
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4413641518