Generating Mouse Line

To introduce a human APOE3 and APOE3Christchurch (APOE3-R136S, E3S/S) cDNA into mouse Apoe genetic locus, a donor plasmid was designed and made. The human APOE3 orAPOE3S/S cDNA and a poly A tail were flanked by left and right homology arms which were designed to insert human APOE3 or APOE3S/S right before the start codon of mouse Apoe gene. Upon recombination, the mouse Apoe promoter and regulatory elements would drive expression of the inserted human APOE3 orAPOE3S/S cDNA, whereas the expression of mouse Apoe gene would be inactivated. A single guide RNA (sgRNA) was selected. RNP containing sgRNA and Cas9 protein was prepared. Pronuclear injections of RNP and the donor plasmid was performed at the Rockefeller University transgenic core facility. Genomic DNA from founders was isolated from tail lysate. To screen the specific knockin mice, two PCR amplifications were performed with primers flanking the outside of the homology arms and on the human APOE3 transgene. The PCR products were sequenced to validate the correct insertion and the locus integrity. Non-specific integration of the donor DNA was characterized by two simple PCR amplifications using two sets of primers on the backbone of the plasmids.

To distinguish homozygous from heterozygous mice, tail DNA from F2 offspring was further characterized by PCR with three primers, two primers on the targeting genomic sequences and one primer on the transgene. Three independent APOE3 or APOE3S/S lines were generated. One APOE3 or APOE3S/S line was used to cross with a transgenic mouse model overexpressing human tau harboring the P301S mutation to evaluate the effect of APOE3 Christchurch on tau pathology. Mice were maintained in barrier facility with 12-hour light, 12-hour dark cycle and stable temperature conditions.