This Il1rap^ex3KO/hAbeta/APOE4/Trem2*R47H quadruple mutant line was generated by utilizing CRISPR/Cas9 endonuclease-mediated genome editing of the Il1rap gene in C57BL/6J-congenic Apoe^{tm1.1(APOE*4)Adiuj} App^em1Adiuj Trem2^em1Adiuj mice (available as Stock No. 030670).
Stock No. 030670 was generated by utilizing CRISPR/Cas9 endonuclease-mediated genome editing of the App gene in C57BL/6J-congenic Apoe^{tm1.1(APOE*4)Adiuj} Trem2^em1Adiuj mice (available as Stock No. 028709).

For the Apoe^{tm1.1(APOE*4)Adiuj} allele (APOE4; available as Stock No. 027894): A targeting vector containing a 3' FRT flanked neo cassette was used to replace exons 2, 3 and most of exon 4 with 1.5 kb of human APOE gene sequence including exons 2, 3 and 4 (and some 3' UTR sequence). The human ApoE sequence also contains a 2 nucleotide A to T substitution in intron 3 for identification of the targeted allele by Southern analysis. The construct was electroporated into C57BL/6J-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission by crossing to C57BL/6J. Heterozygous animals were crossed subsequently to FLP recombinase expressing mice (see Stock No. 005703) to remove the FRT site flanked PGK-neo cassette. Mice that no longer contained the FRT flanked PGK-neo cassette were then backcrossed to C57BL/6J at least once to remove the FLP recombinase transgene.

For the Trem2^em1Adiuj allele (Trem2*R47H; available as Stock No. 027918): Plasmids encoding a single guide RNA designed to introduce a R47H point mutation, with two silent mutations, into the Trem2 gene and the cas9 nuclease were introduced into the cytoplasm of C57BL/6J-derived fertilized eggs with well-recognized pronuclei. Correctly targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by sequencing and PCR and further bred to C57BL/6J (Stock No. 000664) to develop the colony.

For the App^em1Adiuj allele (hAbeta): Plasmids encoding a single guide RNA designed to introduce the G601R, F606Y, and R609H point mutations into the App gene and the cas9 nuclease were introduced into the cytoplasm of Stock No. 028709-derived fertilized eggs with well recognized pronuclei. G601R corresponds to a GGA to CGA nucleotide change, F606Y represents a TTT to TAT nucleotide mutation, and R609H incorporates a CGC to CAT nucleotide change. Correctly targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by sequencing and PCR and further bred to Stock No. 028709 to develop the colony.
Important note about App isoforms and exon numbering: The App-201 isoform (695 aa protein) is encoded by 16 exons, of which the Abeta sequence is encoded by exon 14. The App-206 isoform (770 aa protein) is encoded by 18 exons, of which the Abeta sequence is encoded by exon 16.

For the Il1rap^em2Adiuj allele (Il1rap^ex3KO): Plasmids encoding a single guide RNA designed to delete exon 3 of the Il1rap locus and the cas9 nuclease were introduced into the cytoplasm of Stock No. 030670-derived fertilized eggs with well recognized pronuclei. Correctly targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by sequencing and PCR and further bred to Stock No. 030670 to develop the colony.