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. 2013 Mar 7;8(3):e58034. doi: 10.1371/journal.pone.0058034

Figure 1. Generation of Pyr6-5 Knockouts.

Figure 1

(A) Schematic representation of the generation of Orotidine Monophosphate Decarboxylase (OMPDCase) knockout. The first step is replacement of one PYR6-5 allele with a construct containing both positive selection marker hygromycin phosphotransferase (HYG) and negative marker Herpes simplex virus thymidine kinase (HSVTK) between 34-bp loxP elements. The second step creates the homozygous PYR6-5 −/− through drug pressure with 5-fluoroorotic acid (5 FOA), causing loss of heterozygosity. (B) PCR analysis of the PYR6-5 gene, generating an 870 bp amplicon as described in the Materials and Methods section confirmed the absence of the gene in Pyr6-5 −/− (lane 1) and its continued presence in Pyr6-5 +/− (lane 2). Lane 3 is the control with WT s427 DNA. (C) Southern blot confirming knockout strategy, using probes for the PYR6-5 locus and for the HYG-HSVTK cassette. Lane 1, Pyr6-5 −/−, lane 2, Pyr6-5 +/−, Lane 3, WT s427. Band ‘a’ is PYR6-5, band ‘b’ is HYG-HSVTK.