Figure 5. Generation of Gm114^−/− mice.

(A) Construct design to generate a conditional allele of Gm114. Two loxP sites (yellow triangles) were targeted into the Gm114 locus flanking exons 5 and 6 (green boxes) through homologous recombination. A neomycin cassette (blue box) flanked by frt sites (black triangles), and a thymidine kinase cassette (brown box) were used for positive and negative selection in ES cells. Southern blot and PCR analyses were used to identify targeted ES clones (data not shown). Southern blot analysis was performed using a BamHI restriction digest (cutting sites marked by B) and a specific DNA probe which is indicated by a line and asterisk underneath the targeted allele. (A, B) loxP recombination results in loss of 950bp of the coding region and generates a stop codon (red arrow) at beginning of exon 7 blocking translation of remaining exons. The DNA sequence in exon 7 and its encoded amino acid residues after the frame shift are shown in the box.