Strategy for the generation and characterization of PbALASKO and PbFCKO.

(A) Double crossover recombination strategy to generate PbALAS and PbFC KOs. (B,C) Genomic DNA-PCR analysis indicating the targeted deletion of ALAS and FC sequences in the KOs. (D,E) RT-PCR analysis indicating absence of mRNAs for ALAS and FC in the KOs. (F,G) Southern analysis of DNA from PbWT, PbALAS and PbFC KOs. For PbALASKO confirmation, respective genomic DNA and transgenic plasmid (TP) were digested with BglII and hybridized with 3′UTR specific probe. For PbFCKO, digestion was carried out with SphI and BspDI. Transgenic plasmids were included to rule out the presence of episomes. (H,I) Northern analysis indicating the absence of mRNAs for ALAS and FC in the KOs. (J) Northern analysis for PBGD in the PbALAS and PbFC 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 PbWT and PbKOs giving positive signal (control).