Generation of transgenic <i>R26-AKT1</i>^<i>E17K</i> mouse.

<p><b>A.</b> Schematic representation of the targeting construct used for the conditional knock-in in the <i>R26</i> locus. The human <i>AKT1</i>^<i>E17K</i> cDNA preceded by a loxP-flanked transcriptional stop cassette, was recombined into the R26 locus. Cre-mediated removal of the stop cassette links Rosa26 exon 1 to the exogenous cDNA allowing expression of the transgene. <b>B.</b> Southern blot of EcoRV digested genomic DNA derived from mESCs transfected with the targeting construct carrying mutant AKT1. Lane 1: DNA from non-targeted mESCs, lane 2 and 3: DNA from DNA from two different <i>R26-AKT1</i>^<i>E17K</i> mESCs strains. Endogenous allele corresponds to the 11.5 kb band (<i>WT</i>); mutant allele corresponds to the 4.3 kb band (<i>REC</i>). <b>C.</b> Relative mRNA expression of human AKT1^E17K by Q-RT-PCR in targeted mESCs and in the corresponding cells transfected with the Flp (pFlpE-IRES-Puro) or Cre recombinase (pCre-IRES-Puro). Data are from replicate experiments as the mean±SD. ***p<0.001. <b>D</b>. Genotype analysis by PCR on tail-tip DNA of genetically modified mice as indicated.