De novo production of new knockout mouse lines for the next phase of the KOMP2 Project UM1OD023221 Administrative Supplement August 1, 2021 – July 31, 2022 ABSTRACT The aim of this administrative supplement request is to generate 25 new homozygous viable gene knockout mouse lines ready for production of male and female cohorts for phenotyping in a forthcoming KOMP2-Phase3 project. To do so, this supplement will fund production activities under 3 specific aims. Specific Aim 1 involves gene selection and exdel KO allele design, CRISPR guide in vitro quality control (QC), zygote electroporation, embryo transfer, gestation and parturition, founder (F0) screening by gene specific PCR genotyping, weaning and housing F0 mice until breeding age, F0 breeding and sequence quality control (QC) to confirm germline transmission (GLT) for 25 genes (18 at UC Davis, 7 at TCP). Specific Aim 2 involves QC of N1 mice, production reporting to GenTar, and line expansion of N2 mice to breeding age which will be ready to generate male and female cohorts for phenotyping. Specific Aim 3 involves sperm cryopreservation of 2 heterozygote males for each line produced and deposition into the MMRRC at UC Davis. This work will be completed by July 31, 2022.