Diphtheria Toxin

Lyophilized diphtheria toxin (DTx) from Corynebacterium diphtheriae (1 mg; EMD Millipore) was resuspended in filter-sterilized PBS and stored at −80 °C. Mice were injected intraperitoneally with 1 µg of DTx in 50 µL PBS, or with PBS alone, for two consecutive days prior to infection with B. burgdorferi. Groups consisted of BALB/c DEREG mice administered DTx in PBS with or without subsequent infection, BALB/c DEREG mice administered PBS prior to infection, and wild-type BALB/c mice administered DTx in PBS prior to infection. Mice were weighed immediately before administration of DTx and then regularly thereafter for the duration of the studies. Mice were anesthetized with isoflurane prior to all injections.

The mouse Tip60 genomic fragment was kindly provided by Dr. John Lough at Wisconsin University. Approximately 1.3 and 1.7 kb of the genomic region of Tip60 were inserted into pBluescript SK containing Pgk1-Neo for positive selection and diphtheria toxin A for negative selection (KO II vector) [98 (link),99 (link)]. A loxP was inserted between exons 9 and 10, and an Frt–Pgk1-Neo–Frt–loxP cassette was inserted between exons 11 and 12 to frank exons containing a part of the MYST domain, including the acetyl-CoA binding site with loxP sequences (Figure S1A). The linearized targeting construct was electroporated into E14tg2a embryonic stem (ES) cells, which are derived from 129/Ola strain mice. ES cell clones were selected in ES cell culture media supplemented with 200 µg/mL of G418. Targeted clones were screened by Southern blot analysis using 5′ and 3′ probes (Figure S1). Three of the correctly targeted clones were expanded and injected into C57BL/6J blastocysts. Chimeric males were bred to C57BL/6J females to obtain heterozygous Tip60-targeted mice. In removing the Pgk1-Neo cassette, pCAGGS-FLPe was injected into one-cell stage embryos derived from C57BL/6J females mated with heterozygous Tip60 targeted males, and the embryos were transplanted into the oviducts in ICR mice. The removal of the Pgk1-Neo cassette was confirmed by PCR using mouse tail DNA. This mouse line, which contains a floxed Tip60 allele without a Pgk1-Neo cassette (F allele), was used in this study. The Tip60^F/+ mouse was backcrossed to the C57BL/6 mouse at least 6 times.
In order to delete Tip60 from NSCs, Nestin-Cre⁺ (Nes-Cre⁺) mice, in which Cre recombinase is expressed in NSCs after embryonic day 11.5 (E11.5), were crossed with Tip60^F/+ mice. Homozygous floxed Tip60 females (Tip60^F/F) were crossed with Tip60^F/+;Nes-Cre⁺ males to produce mice, in which the Tip60 genes were knocked out in the NSCs (Tip60^F/F;Nes-Cre⁺ indicated as Tip60 cKO hereafter). All genotypes were confirmed by PCR using primers listed in Supplementary Table S1. All experiments were performed using Tip60 cKO mice with their littermates as controls. The mice were maintained under controlled environmental conditions consisting of a 12 h/12 h light and dark cycle with free access to standard mouse chow and water.