Scientific MADLIBS

Randy has requested a scientific blog next so I’ll explain my BIG FAT experiment in 20 sentences.

--Names and places have been changed to protect the innocent.

1. I am conducting pre-clinical testing of vaccinations for the prevention of escape mutant induced MOMMY resistance. 2. There are three mutations I am going to vaccinate against, THEO, AIDAN, and JOEL point mutations. 3. The THEO mutation changes the P-loop domain of the MEGAN kinase on KARA so that MOMMY no longer binds. 4. The AIDAN mutation is the worstest mutation due to the fact that it not only confers resistance to MOMMY but also to all second and third generation small molecule KARA kinase inhibitors that have been developed to date. 5. The JOEL mutation changes the amino acid in the LEANNE domain necessary for self-inhibitory binding to the PONCHO therefore preventing the KARA protein from taking on the inactive state required for MOMMY binding. 6. The vaccine will eventually consist of about 400 amino acids of the JASON, LEANNE and PONCHO of KARA that has been mutated to encode the three MOMMY escape mutations mentioned above. 7. The human and mouse MEGAN orthologs of this region differ at two amino acids which must be mutated to conform to the mouse ortholog in order to prevent confounding immunization in mice against the human and thus foreign form of the protein. 8. I have succeeded in subcloning the JASON, LEANNE, PONCHO of KARA from the MSCV expression vector into the smaller pBlueScript vector for QuikChange mutagenesis. 9. Each of the five necessary mutations has been produced and sequential mutatgenesis is underway to create a construct containing all five mutations. 10. Once this has been accomplished the construct will be sent to CHRISTINA for production of the yeast-based vaccine, which has been dubbed RANDY for RANDY. 11. CHRISTINA calls their yeast-based vaccines BEBO. 12. Until this vaccine is made I am using peptide based vaccines to determine whether mice possess functional T cells specific for any of the three escape mutant epitopes we are interested in. 13. So far I have immunized 28 mice with either of two AIDAN nine amino acid peptides or a MELANIE junctional (JCN) control. 14. Several of the mice died after the booster injection of peptide for reasons that are under investigation. 15. Some of the remaining mice are currently being challenged with either target peptide or irrelevant peptide in vivo. 16. The results for these CTL assays will be available tomorrow afternoon. 17. T cells from the remainder of the mice will be assayed for epitope-specific cytolytic activity in vitro on Monday. 18. QuikChange mutatgenesis is also being conducted to produce expression vectors carrying the escape mutations so that leukemic cells carrying KARA MOMMY escape mutations can be used as target cells in CTL assays. 19. Kavita is my rotation student and has assisted with many of these experiments. 20. She also does not wish harm to the little mousies and would probably have used the sticky paper but wouldn’t have suffocated them cause she can barely touch one without jumping 12 feet so I have to catch them all for her and do anything that has to do with live little mousies. –The End