Extended Data Fig. 2: Generating and characterizing knock-in mice with ACE expression restricted to microglia. | Nature Aging

Extended Data Fig. 2: Generating and characterizing knock-in mice with ACE expression restricted to microglia.

From: Boosting angiotensin-converting enzyme (ACE) in microglia protects against Alzheimer’s disease in 5xFAD mice

Extended Data Fig. 2

a, Rosa-hACE-flox mice were generated by targeting the Gt(Rosa) safe harbor locus in mouse embryonic stem cells. The targeting construct contains a loxP-site flanked transcriptional stop sequence upstream of a bicistronic TurboGFP and c-terminal flag-tagged human ACE (hACE-3xF) expression unit that is expressed after loxP site recombination in the genome. Embryonic stem cell clones were screened by PCR (3′ screen) to identify positive clones that generated a 4,355 bp PCR amplicon. Secondary screening was used to detect 5’ homologous recombination by Southern blotting on MfeI restricted genomic DNA to identify a 10.3 kb fragment indicative of heterozygous homologous recombination and germline heterozygous ACE-flox (Rosa+/hACE-flox) mice were generated. b, ACE-flox mice were mated to Cx3Cr1+/CreERT2 mice to generate RACE- (Cx3Cr1+/CreERT2; Rosa+/+) and RACE+ (Cx3Cr1+/CreERT2; Rosa+/hACE-flox) mice. c, RACE- and RACE+ mice were injected with Tamoxifen (TMX; 125 mg/kg, IP) for four consecutive days to activate Cre-recombinase in R- and R+ myeloid-derived cells. d, Myeloid cells isolated from brain, spleen and blood were gated on DAPI, CD11b and Cx3Cr1 and then sub-gated on GFP to detect transgene expression, 7, 21 and 180 days after TMX injection. e, Transgene expression persisted in myeloid cells in the brain and became undetectable in the blood and spleen by 3 weeks after TMX injection (data are plotted as mean ± SEM).

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