Construction of the targeting vector and generation of Ptdsr-knockout and gene-trap mice

Targeting vector
A Ptdsr-containing bacterial artificial chromosome (BAC) clone (GenBank accession number AC091694; RP-23-316F3) was isolated by sequence homology from a C57BL/6J genomic BAC library (RP-23; BACPAC Resources, Oakland, USA). A 14.5 kb KpnI/BamHI fragment containing the entire Ptdsr locus and 5' and 3' flanking regions was subcloned from this BAC clone and a 1.9 kb RsrII/AatII fragment containing exons I and II of the Ptdsr gene was replaced by a 1.2 kb loxP-flanked neomycin-resistance gene cassette (neo).

Homologous recombination in ES cells and generation of germ-line chimeras
Bruce4 ES cells were transfected with KpnI-linearized targeting vector and selected with G418. ES-cell clones resistant to G418 were isolated and analyzed by Southern blot analysis for homologous recombination events within the Ptdsr locus. Chimeric mice were produced by microinjection of two independent homologous recombinant (Ptdsr+/-) ES cells into BALB/c blastocysts and transfer to pseudopregnant foster mothers followed by development to term. Chimeric males were mated with C57BL/6J females. From the two selected ES-cell clones, one successfully contributed to the germ-line. Germ-line transmission of the mutant allele was verified by PCR and Southern blot of genomic DNA from black coat-color F1 offspring.

Ptdsr gene-trap and generation of germ-line chimeras
An ES-cell line carrying a β-geo gene-trap vector in the Ptdsr locus was identified by searching the BayGenomics database (BayGenomics, San Francisco, USA; [48]) with the full-length Ptdsr cDNA. A single ES-cell line was identified carrying the gene-trap in intron V, between exons V and VI of the Ptdsr gene. Chimeric mice were generated by microinjection into CB20 blastocysts and transfer to pseudopregnant foster mothers. Chimeric males were mated with 129P2/OlaHsd females. Germ-line transmission of the mutant gene-trap allele was verified by expression analysis using β-galactosidase staining and RT-PCR.