Strategy for insertion of the EGFP gene into thelys locus.

(A) A schematic representation of the linearized targeting construct and the genomic locus before and after recombination. The 4 exons of the lys gene and the probe used are shown as solid black boxes. The transcriptional direction of lysozyme is indicated by an arrow, theEGFP gene as a shaded box, and the LoxP-sites as boxes containing “less than” signs. The Tk-neo cassette, with its transcriptional direction (arrow), is indicated with an open box, and the HSV-Tk genes, with their transcriptional directions (arrows), are indicated with hatched boxes. After linearization, the targeting construct was either left untreated, nonsplinked (B), or splinked (C) by ligating to oligonucleotides forming hairpin “splinkers” in order to protect the fragment ends from exonucleolytic degradation. The fragments were then electroporated into R1 ES cells, the cells were cultured in G418 and ganciclovir, and double-resistant clones were isolated. Finally genomic DNA was prepared, digested with EcoRI, and analyzed by Southern blot analysis with the indicated probe. The 9-kb band in panels B and C corresponds to the fragment obtained from wild type clones; the 6-kb band corresponds to the fragment generated by homologous recombination in some clones. (These are indicated with a star).