Generation and validation of conditional, floxed and constitutive knockout (KO) alleles of the H3f3b gene. (A) (top) The wildtype (WT) H3f3b allele. (second row) The targeting vector contains loxP sites (triangles) that flank exons 2 to 4, a 4.3 kb 5’ arm of homology, a 5.3 kb 3’ arm of homology, diphtheria toxin A (DTA) cassette, and a neomycin (Neo) cassette flanked by frt sites (vertical double arrows). The Neo element allows for positive selection in embryonic stem (ES) cells, while the DTA element permits negative selection in ES cells. (third row) After homologous recombination of the conditional knockout construct, the H3f3b gene is expressed until Cre-mediated deletion of exons 2 to 4 (bottom), deleting the entire coding sequence (CDS). (B) Southern blotting of H3f3b WT and floxed mice using a 3’ probe yields the expected 20 kb and 17 kb bands, respectively validating appropriate gene targeting. (C) Southern blotting using a 5’ probe generates the predicted 20 and 10 kb bands for KO and WT alleles in the samples of the indicated genotypes. With this probe a background band (*) was present in all samples. (D) Mapped reads from ChIP-Seq assays on KO and WT mouse embryonic fibroblasts (MEFs) 1 and 2 for histone 3 lysine 4 tri-methylation (H3K4me3) and histone 3 lysine 9 acetylation (H3K9ac) indicate precise deletion of the H3f3b floxed region with undeleted Exon 1 still exhibiting histone marks. (E, F) qPCR of H3f3b (E) and H3f3a (F) mRNA levels in MEFs of indicated genotypes. (G) qPCR assay of H3f3b mRNA levels in conditional KO MEFs. Error bars are standard deviations. ND = none detected.