Fig 1.
Gene-targeting at the leu1 locus in fission yeast.
(A) Schematic representation of the leu1 knockout construct. A BamHI/ClaI fragment containing the leu1 ORF was cloned in a pBlueScript vector and a ura4 marker gene was inserted in the HindIII site in the middle of the leu1 ORF to generate a leu1 knockout construct. The resultant knockout vector was digested at the edges of both homologous arms using BamHI and ClaI (left), or digested in the pBlueScript vector using PvuII (Right). (B) A digested knockout vector (5 μg) was transformed into a fission yeast strain (h+ ura4-D18). The number of transformants selected for uracil prototrophy, percentage of clones showing leucine auxotrophy, and the estimated number of gene-targeting events per transformation are shown.
Fig 2.
Gene-targeting at the POLD1 locus in DT40 cells.
(A) Schematic representation of the POLD1 knockout construct. A PstI fragment containing a part of the POLD1 locus was cloned in a pBlueScript vector and a hisD-resistant marker gene was inserted in the NdeI site in the middle of the POLD1 fragment to generate a POLD1 knockout construct. The resultant knockout vector was digested at the edges of both homologous arms using PstI (left), or digested in the pBlueScript vector using PvuII (Right). (B) The digested knockout vector (50 μg) was transfected into DT40 cells (#653 wild-type cl-18). The number of colonies selected for resistance to L-histidinol, percentage of colonies in which the POLD1 gene was disrupted, and the estimated number of gene-targeting events per transfection are shown. (C) Representative image showing Southern blot for assessing pold1 gene disruption. Due to significant difference of the pold1 gene locus (around exon 10) between dad and mom allele in DT40, wild type cells exhibited two bands.
Fig 3.
Efficient crossover-mediated homologous integration in DT40 cells.
(A) Schematic representation of homologous integration using gapped plasmids. An homologous-arm sequence (purple bar) was cloned in a vector carrying a puromycin-resistant gene (red bar). The resulting vector was digested with a restriction enzyme to make the gap in the homologous sequence. Gapped plasmids were transfected into DT40 cells and homologous integration accompanied by chromosomal crossover resulted in the integration of whole plasmids into the homologous region. Targeted integration was assessed by PCR using primers (arrows). (B) Length of the homologous arm and percentage of the homologous targeting for integration in stathmin and vimentin loci. (C) Representative image showing PCR products using the primers shown in A. λ-phage DNA digested with EcoT14I was used as a size maker.
Fig 4.
Flip-in system to fuse epitope tags to proteins of interest.
(A) Schematic representation of the flip-in system to fuse epitope tags to proteins of interest. Genomic sequence containing the last exon but lacking the stop codon (purple bar) was cloned and connected with an in-frame sequence tag. The resulting targeting vector was digested in the middle of the cloned homologous arm (purple bar). Gapped plasmids were transfected. At least for 10 days the transfected cells were cultured before assessing homologous integration. Homologous integration resulted in the replacement of the original gene by the modified gene encoding the tagged protein. (B) The percentage of homologous integration for tagging OTU6B, histone H1-like protein, histone H1.03, and RPA was summarized. (C) Representative image of western blot with anti-FLAG antibody showing FLAG-tagged histone H1.03. (D) Dot-plot representation of flow-cytometric analysis to evaluate fluorescent emissions (green) from RPA-GFP. The cell was stained with propidium iodide (red) to exclude the dead-cell fraction. Strength of green and red fluorescence was plotted on the x-axis and y-axis, respectively, in a logarithmic scale.
Fig 5.
Flip-in system to stably express transgenes of interest.
(A) Schematic representation of a flip-in system to stably express transgenes. A genomic sequence containing a 2.8 kb fragment of an OVA gene (purple bar) was cloned and connected with a puromycin-resistant gene and an expression cassette (β-actin promoter, multi-cloning site and polyadenylation signal). The resulting targeting vector was digested with KpnI and transfected. At least for 10 days the transfected cells were cultured before assessing homologous integration. Homologous integration results in the integration of the expression cassette into the OVA locus. Target integration was assessed by PCR using primers (arrows). (B) Representative image showing PCR products using the primers shown in A. λ-phage DNA digested with EcoT14I was used as a size maker. (C) Complementation of camptothecin hypersensitivity of tdp1-/- cells by stable expression of human TDP1 cDNA. Cellular sensitivity to camptothecin was analyzed. Survival rate was calculated as the percentage of surviving cells treated with DNA-damaging agents relative to untreated surviving cells. Error bars show the SD of mean for three independent assays. The concentration is displayed on the horizontal axis, while the survival rate is displayed on the y-axis on a logarithmic scale.