Generation of Olig1 null mouse lines. (A) The Olig1 ORF was replaced by Notch1 intracellular domain (NICD) by homologous recombination in ES cells followed by blastocyst injection to generate Olig1(-/-) mice. A Pgk-Neo cassette, flanked by loxP sites, was inserted just upstream of NICD. Arrows indicate the 5’-3’ directions of each coding region. (B) Southern blot of NcoI-digested genomic DNA using a 700 bp 5’ probe revealed a 4.1 kb band in wild type mice. An additional 4.9 kb band was identified in heterozygous mice. (C) Southern blots of NcoI/AscI-digested genomic DNA using a 200 bp 3’ probe revealed a 5.3 kb band in wild type mice. An additional 5 kb band was identified in heterozygous mice due to the introduction of a new AscI site in the targeting vector as part of the cloning procedure. (D) Olig2 expression was not up-regulated in Olig1(-/-) mice. Quantitative PCR using cDNA templates acquired from E13.5 and E18.5 forebrain tissues revealed that Olig1 heterozygotes and homozygous knockout mice expressed similar amounts of Olig2 mRNA. (E) As an alternative approach to generating Olig1 null mice, Olig2 PAC transgenic mice (generated by pronuclear injection; ) were crossed to Olig1/2 double KO mice to rescue Olig2 and produce [Olig(-/-), Olig2(Tg)] mice, which phenocopy Olig1(-/-). Arrows show the direction of transcription.