Combined with genetic etiological models in mice, such cell type-based approaches may further contribute to understanding the genetic architecture selleck screening library and pathogenic mechanisms of neurodevelopmental and psychiatric disorders. Gene targeting vectors were generated using BAC recombineering (Lee et al., 2001) and, in a few cases, PCR-based cloning approach (Figure S1). For constitutive Cre lines, either an ires-Cre cassette was inserted immediately after the STOP codon or a 2A-Cre cassette was inserted in frame just before the STOP codon of the targeted gene. For inducible lines, CreER was inserted at the translation initiation site

of the targeted gene. If the ATG codon of the targeted gene is in the first coding exon, PD0332991 order a CreER-intron-polyA cassette was used; if the ATG codon is not in the first coding exon, a CreER-polyA cassette was used. Two to five kb upstream or downstream regions of the targeted loci were cloned into targeting vector as 5′ and 3′ homologous arms, respectively ( Table 1). All targeting constructs include an frt-Neo-frt cassette and

a tyrosine kinase cassette or Diphtheria toxin cassette for positive and negative selection in ES cells, respectively. Detailed information on targeting constructs for each line is available at http://www.credriver.org. For each gene of interest, two partially overlapping BAC clones from the RPCI-23&24 library (made from C57BL/b mice) were chosen from the Mouse Genome Brower. BAC DNA was transferred from DH10B strain to SW105 strain by electroporation. The

identity and Oxygenase integrity of these BAC clones were verified by a panel of PCR primers and restriction digestions. We constructed a series of “building vectors” containing the essential elements for different strategies of BAC targeting (Table S1; Figure S1A). These elements were inserted into P451B (gift of Dr. Pentao Liu), a modified version of PL451 without a loxP site (Liu et al., 2003) in front of the frt-Neo-frt cassette. The Neo gene is driven by both the PGK promoter for G418 selection in ES cells and the EM7 promoter for Kan selection in Escherichia coli. A BAC targeting vector was generated for each gene by cloning appropriate 5′ and 3′ homology arms from the gene into a building vector, flanking the CreERT2frt-Neo-frt cassette. For targeting to the ATG initiation codon, we typically use 300–500 bp DNA fragments immediate upstream and shortly downstream for 5′ and 3′ homology arms, respectively. We used the PL253 retrieval vector (Liu et al., 2003) as the backbone of our knockin vectors (Figure S1B). PL253 contains the HSV-TK gene driven by the MC1 promoter for negative selection in ES cells. This cassette is flanked by multicloning sites. Knockin cassette was retrieved from the modified BAC clones into PL253 by recombineering.