Regulatory Mechanisms of Glycoprotein Sialylation

The Whiteheart laboratory’s primary role in this proposed research is the generation, maintenance, validation and initial use of the mouse strains listed below. All animal procedures done at University of Kentucky are approved by the local IACUC (protocol number 2019-3384). The estimated numbers of animals per strain is 30: • VAMP3 knockout • VAMP2/3 double knockout • VAMP2/3/8 triple knockout • Munc13-4 knockout • IKKß knockout • Tomosyn-1 knockout • NBEALl2 knockout • WT control As detailed in Specific Aim 2 and within the Whiteheart laboratory, platelets samples will be prepared from the strains above and sent to the Cobb lab for analysis. Additionally, mice will be immunized, receive adoptive cell transfers, and be used for harvesting megakaryocytes, platelets and plasma for analyses at the Cobb laboratory. Platelets will also be used ex vivo to create thrombin-induced ‘releasates’ for analysis. Super Resolution (3DSIM) fluorescence microscopy will be performed on platelets to localize IgG and ST6Gal1 in selected mutant mice. All IgG and glycomic analyses will be performed by the Cobb laboratory. Finally, sperm from selected mutant mice with interesting and robust IgG glycosylation phenotypes (TBD) prepared and sent to the Case Western Reserve University Animal Resource Center (CWRU ARC) for rederivation of the strains at CWRU for so that the lines can be crossed onto the CWRU-local strains – specifically the PcKO and PF4-DTR mice.