C57bl 6j female

Caveolin-1 transgenic (Cav1^Tg+) mice were
obtained from Dr. Ferruccio Galbiati at the University of Pittsburgh with
the kind permission of Dr. Michael Lisanti (12 (link)). Male heterozygous transgenic mice were bred to C57BL6/J
females (Jackson Labs; Stock no. 662). Tg+ and Tg−
littermates were used to generate cohorts for analysis.

Chimeric mice were produced by microinjection of independent mIrf2⁺/− ES cell clones into E3.5 C57BL/6J blastocysts and transferred to pseudopregnant foster mothers. Chimeric males were mated with C57Bl/6J females (Jackson Laboratory). Germ line transmission of the mutant allele was confirmed by PCR analysis of tail DNA from mice with an agouti coat color. The PGK-Neo cassette was removed from mouse Irf2^+/− mice by crossing with Flp-deleter mice (Jackson Laboratory stock #009086) (Farley et al., 2000 (link)) and PCR genotyping and sequence validation of recombination at the FRT sites.

8-week-old C57BL/6J females (Jackson Laboratory) were used. All mouse experiments were reviewed and approved by the Institutional Animal Care and Use Committee of New York University Langone Health (NYULH). All experiments were performed according to NIH guidelines, the Animal Welfare Act, and U.S. federal law. Wild type or H526Y (MG1655) cells were sub-cultured at a 1:100 dilution in LB from an overnight culture of 3 single colonies for 3 hours. The bacteria were washed 2 times with PBS and then O.D. normalized to ~1 x 10⁹ CFU/ml in PBS. 8-week-old female C57BL/6J (Jackson Laboratory) were infected intraperitoneally with 3 x 10⁸ CFU. 1-hour post infection, the mice were treated with either PBS + 10% DMSO or 20 mg/kg A22 by intraperitoneal injection. 1-day post infection, the mice were sacrificed by CO₂ inhalation. Peritoneal lavages were performed using 3ml PBS to rinse the peritoneal cavity. Other organs were homogenized in 1 ml PBS. Statistical significances were calculated using one-way ANOVA with Kruskal-Wallis test to correct for multiple comparisons.

A single male that was confirmed to be heterozygous for the mutation and free of off-target effects was chosen as the founder for line generation. He was mated with wildtype C57BL/6J females purchased from Jackson Laboratories (Bar Harbor, ME) and all F1 mice were genotyped by both PCR and restriction digest as well as confirmatory Sanger sequencing to establish the MAP2 S/E line. Heterozygotes were backcrossed into purchased wildtype C57BL/6J strain to produce animals for all experimental studies mitigating against genetic drift of the line. Throughtout the text animals are referred to as S1782E mice.
Animals were identified with metal ear tags and tail snip DNA samples were obtained for genotyping by PCR prior to weaning at approximately postnatal day 21. Genotypes of all mice included in studies were confirmed following sacrifice. Both male and female animals were included in all studies. Animals were under specific pathogen free conditions and housed in standard microisolator caging (Allentown Caging Equipment, Allentown, NJ, USA) in groups of up to 4 males or 5 females, maintained on a 12-h light/dark cycle (lights on at 7 AM), and were provided with food and water ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh.