Figure 2.

Generation of Atrx gene-targeted mice. (A) A 6.2-kb SstI fragment of the Atrx gene was used to engineer a targeting construct containing a floxed Neo^r cassette within intron 17 and loxP sites flanking exon 18. Two herpes simplex virus thymidine kinase (HSV TK) genes were included to allow for positive and negative selection of recombinant ES cell clones. Female mice homozygous for the floxed Atrx allele (Atrxlox^P) were bred to heterozygous Foxg1Cre male mice to generate KO male mice, referred to as AtrxFoxg1Cre. (B) Southern blot analysis of genomic DNA isolated from the cortices of newborn pups of different genotypes. F/Y, floxed-ATRX X chromosome; F/WT, wild-type X chromosome; Cre⁺, Cre-positive; Cre^–, Cre-negative. Right margin: F, floxed allele; R, recombined allele. (C) RT-PCR analysis of RNA isolated from newborn forebrains of AtrxFoxg1Cre males (F/Y, Cre +), control littermates (F/Y, Cre –), or heterozygous females (F/WT) with or without Cre. Fragments were amplified with Atrx primers 17F and 20R, as indicated in A, or Gapdh primers (lower band) in control reactions. As expected, the amplified product was shorter when Cre was present due to recombination of loxP sites. (D) Western blot analysis of proteins isolated from newborn forebrains of wild-type, heterozygous, or knockout mice using an antibody that recognizes both full-length ATRX protein and the truncated isoform ATRXt (39f) or a C-terminal antibody (H300) that detects only the full-length isoform. Loss of full-length ATRX expression is observed in knockout animals, while ATRXt expression is not affected. Tubulin was used as a loading control.