Effects of the Sox17mut5 mutation on SOX17 function in mouse embryonic gallbladder development
Effects of the Sox17mut5 mutation on SOX17 function in mouse embryonic gallbladder development
Pups were generated by crossing an 8 week old heteozygous male mouse with a heterozygous 6-8 week old female mouse. The date of birth for the pups was denoted as P0. Gallbladders from P21 Sox17mut5/mut5 or Sox17+/+ were isolated at P21 and placed into ice cold PBS.   Sox17mut5 (Sox17em5Mgn) mutant allele was generated using CRISPR/Cas9 mutagenesis by pronuclear injection into  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.