Figure 1.

(A) Targeted disruption of the mouse GRP94 gene. Scheme of the murine gene and the targeting vector. The 18 exons of the GRP94 gene (black boxes) and the introns (thin lines, lengths determined by exon primer PCR and/or sequencing analysis) are drawn to scale, with a gap between exons 11 and 18. The targeting vector contains 1.2-kb 5′ homology generated by PCR amplification and 8.0-kb 3′ homology in an EcoRV fragment. The neo resistance cassette interrupts the coding region at the end of exon 3, 61 amino acids into the mature protein. Its transcriptional orientation is opposite that of the GRP94 gene, as marked by the arrow. The 5′ homology region is flanked by tk, the herpes virus thymidine kinase gene used for negative selection. (B) Correct targeting in two mice was determined by Southern blotting with probe A, located 5′ of the insertion (see arrow in A), after digestion with HindIII (H) or EcoRI (R). A new 6.8-kb EcoRI fragment and a new 6.1-kb HindIII fragment are present in the genome of the correctly targeted mice.