Strategy for the generation and characterization of <i>Pb</i>ALASKO and <i>Pb</i>FCKO.

<p>(A) Double crossover recombination strategy to generate <i>Pb</i>ALAS and <i>Pb</i>FC KOs. (B,C) Genomic DNA-PCR analysis indicating the targeted deletion of <i>ALAS</i> and <i>FC</i> sequences in the KOs. (D,E) RT-PCR analysis indicating absence of mRNAs for <i>ALAS</i> and <i>FC</i> in the KOs. (F,G) Southern analysis of DNA from <i>Pb</i>WT, <i>Pb</i>ALAS and <i>Pb</i>FC KOs. For <i>Pb</i>ALASKO confirmation, respective genomic DNA and transgenic plasmid (TP) were digested with <i>BglII</i> and hybridized with 3′UTR specific probe. For <i>Pb</i>FCKO, digestion was carried out with <i>SphI</i> and <i>BspDI</i>. Transgenic plasmids were included to rule out the presence of episomes. (H,I) Northern analysis indicating the absence of mRNAs for <i>ALAS</i> and <i>FC</i> in the KOs. (J) Northern analysis for <i>PBGD</i> in the <i>Pb</i>ALAS and <i>Pb</i>FC KOs giving positive signals (control). (K,L) Western analysis indicating the absence of ALAS and FC proteins in the KOs. (M) Western analysis for hsp60 in the <i>Pb</i>WT and <i>Pb</i>KOs giving positive signal (control).</p>