Fig. 1. Construction of transgenic lines. (A) Krox20 gene and extragenic regions extending from –31 kb to +40 kb relative to the start site of transcription (upper). Cosmid inserts extending from –31 kb to –2.3 kb and +3 kb to +40 kb as well as the –4.5 kb to +7 kb Krox20/lacZ construct used to generate the –31/+7 Krox20/lacZ and –4.5/+40 Krox20/lacZ transgenic lines (lower). Restriction enzymes used to prepare these fragments for transgenesis and for Southern analysis are shown. Krox20 exons are indicated as solid black rectangles. Solid squares identify vector sequences. (B) Southern analysis of genomic DNA. A lacZ SstI/NdeI fragment was used as a probe. Enzymes used to diagnose recombination are indicated. Four –31/+7 Krox20/lacZ founders that were positive in PCR for both lacZ and cosmid vector sequences were analysed (left panel). DNA from Krox20^lacZ/+ mice was included to identify the size of the recombinant band as the lacZ sequence was inserted at the BglII site in both the knock-in and transgenic mice (Schneider-Maunoury et al., 1993). Three –4.5/+40 Krox20/lacZ transgenic founders positive in PCR for both fragments injected were analysed (right panel). The size control consisted of a Krox20/lacZ plasmid with genomic sequences extending from –4.5 kb to +10.5 kb. The arrowheads indicate the size of bands consistent with recombination, which are 11.7 kb and 8.5 kb for the –31/+7 Krox20/lacZ and –4.5/+40 Krox20/lacZ transgenes, respectively. In both panels DNA from the B6D2 genetic background was included as a negative control.