Generation of <i>EbfmiRNA</i> transgenic murine ES cells.

<p>(A) Schematic representation of the <i>Rosa26</i> wild-type locus. A solid line represents regions involved in the targeting construct, while a dashed line depicts genomic regions beyond the targeting construct. The targeting strategy consists of inserting the transgene into the XbaI site in intron 1, which is flanked by homology arms including exons1, 2 and 3. The transgene consists of the synthetic CAG-promoter, a <i>PGK::Neo</i> cassette with a transcriptional Stop sequence flanked by <i>loxP</i> sites, the RNAi insert as a black box, and a polyA sequence. In the third line the insert is detailed as consisting of the <i>Gfp</i> gene, and DNA sequences encoding for short hairpin mRNA against <i>Ebf3</i>, <i>Ebf2</i> and <i>Ebf1</i>. These sequences are surrounded by the flanking region of miR155, which is recognised by the ribonuclease Drosha. The locus after recombination is depicted in line 4, also indicating the direction of transcription from the CAG promoter and the <i>PGK::Neo-Stop</i> cassette. Endogenous and newly introduced restriction sites are indicated and the resulting fragment lengths for wild-type and targeted allele with specific restriction enzymes are given. Southern blot probes corresponding to the <i>5</i>’ region and to the <i>Gfp</i> gene are indicated. (B) Southern blot of genomic DNA from one wild-type and four mutant ES cell clones digested with <i>Eco</i>RI and <i>Sca</i>I as indicated, and hybridised to the <i>5</i>´ probe (as shown in A). Bands corresponding to the wild-type (wt) and the targeted (int) allele are indicated. (C) Southern blot of genomic DNA as in B, hybridised to the internal <i>Gfp</i> probe. Bands of the correct size appear in all four targeted ES cell clones as indicated.