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. 2018 May 27;285(14):2662–2678. doi: 10.1111/febs.14511

Figure 7.

Figure 7

Assessing the essentiality of G5K. (A) Schematic representation of the gene replacement strategy and possible outcomes. The light blue bars represent the 5′‐ and 3′‐ UTRs flanking the G5K locus used for gene replacement by homologous recombination. A map of the rescue construct pIR1SAT_LdG5K is included showing the Swa I restriction sites used to linearise the construct targeted for insertion into the (ectopic) ribosomal DNA locus. (B–E) Genotypic analysis of transgenic clones for WT, SDR, DDR and their corresponding rescue constructs (RCWT,RCSDR and RCDDR). gDNA samples were digested with Sac II and Southern blots prepared as described in Experimental procedures. Blots were sequentially probed and stripped with the following: LdG5K (B, C); puromycin N‐acetyl transferase (D) and hygromycin phosphotransferase (E). Coloured arrows indicate that gene replacement had occurred at the G5K chromosomal locus. Presence of the rescue construct in WT, SDR and DDR clones is highlighted by a green arrow.