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. 2019 Nov 8;8(11):1406. doi: 10.3390/cells8111406

Figure 2.

Figure 2

Generation of dck1 knockin strains, (A) Schematic representation of the strategy used for the generation of dkc1 knockin strains by homologous recombination. The genomic organization of the dkc1 locus is represented to scale in the upper part indicating the dkc1 gene, the upstream DDB0308306 gene and the intergenic region that contains the putative dkc1 promoter region. The vector used for homologous recombination is represented underneath and includes the blasticidin resistance cassette (BSR) flanked by two Lox sites. Mutated residues are represented as red asterisks on the dkc1 gene. The product of homologous recombination is represented bellow. The location of the oligonucleotides used for verification of homologous recombination is indicated. The Bsr oligonucleotide hybridizes into the blasticidin-resistance cassette while the DKCK5 oligonucleotide correspond to a region of the BBB0308306 genes located upstream of the 5′ arm of the recombination vector. The position of the oligonucleotides used for amplification and sequencing of the mutated region, SeqF and SeqR, is also indicated. The lowest scheme represents the structure of the locus after removal of the BSR cassette upon expression of the Cre recombinase. Only one Lox site and one short region of the cloning vector remain in the intergenic region. (B) DNA was isolated from several clones obtained after homologous recombination using the knockin-1 vector and before BSR cassette removal. The insertion of the BSR cassette in the dkc1 locus was tested by PCR using the oligonucleotides DKCK5 and Bsr, whose location is indicated in Panel A. PCR products were analyzed by agarose gel electrophoresis. The number of each clone is shown in the upper part of the picture. The arrow drown in the lower part of the picture indicates the clone selected for further analysis. The migration of the more relevant fragments of the 1 Kb ladder from Nippon Genetics Europe GMBH (Germany) is shown to the right. (C) DNA from clones obtained after homologous recombination using the knockin-2 vector was analyzed for the insertion of the BSR cassette by PCR as indicated in Panel B. Migration of the 1Kb ladder is shown to the left of the panel.