Effects of the Sox17 ∆50 mutation on SOX17 function in mouse embryonic liver development
Effects of the Sox17 ∆50 mutation on SOX17 function in mouse embryonic liver development
Embryos were generated by crossing a single housed 8 week old heteozygous male mouse with a heterozygous 6-8 week old female mouse. Female mice with vaginal plugs were defined as being E0.5. At the E12.5 time point, embryonic tissues were micro-dissected in cold phosphate buffered saline (PBS) and embryos staged according to Theiler staging criteria. Sox17∆50  (Sox17em3Mgn) mutant allele was generated using CRISPR/Cas9 mutagenesis by pronuclear injection into  fertilized zygotes from the mating of CD-1 mice. Founder animals were backcrossed to wild type CD-1 mice for three generations before interbreeding to produce homozygous mutant mice.    Mice were fed a regular chow  (4.5% fat content, PicoLab, 5L0D) diet. Total RNA was extracted from isolated mouse pancreatic islets using the Maxwell simplyRNA kit (Promega). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. Final library was complete after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.