Table 1.
Primer sequences.
Fig 1.
Schematic of Cas9-mediated homologous recombination targeting vector-mediated genome engineering of human proximal tubule cells at the KIM-1 locus.
Cas9-gRNA mediated DNA break (red lightning bolt) facilitates integration via homology arms. GFP-T2A-Luc is fused in-frame into exon 4. Floxed (red triangles) RFP-T2A-Puro permits selection of targeted cells and the selection cassette can be removed with Cre recombinase, if desired. The RFP-T2A-Puro cassette is flanked by insulator elements (black hexagons). GFP, green fluorescent protein; T2A, self-cleaving peptide sequence; LUC, firefly luciferase; polyA, polyadenylation sequence; EF1a, EF1a constitutive promoter; RFP, red fluorescent protein; Puro, puromycin N-acetyl-transferase.
Fig 2.
Stable transfection and isolation of HK-2 KIM1-reporter clones.
Unmodified cells (A) were transfected with the gene targeting vector and active Cas9 and evaluated for RFP expression on day 2 (B). Puromycin selection permitted isolation of HK-2 clones with 100% of cells expressing RFP (C and D).
Fig 3.
Verification of gene tagging in generating KIM-1-reporter cell lines.
PCR was used to verify proper gene tagging by amplifying the genomic sequence upstream and downstream of the homology arms in the targeting vector with additional primers within the integrated DNA sequence. Correct upstream PCR results in a band of 1621 bp whereas correct downstream amplification results in a band of 1686 bp. 16 clones were evaluated as well as mock transfected (M) and untransfected (U) cells. Clones 5 and 9 (black arrows) were chosen for further studies due to sequence verified in-frame gene tagging.
Fig 4.
KIM-1 reporter cell line response to hypoxia.
(A), clones 5 and 9 demonstrate increased luciferase expression via IVIS imaging in response to hypoxia (1% O2) over a course of 24–48 hours. (B), This hypoxic response was reproducible with little variation between clones. Cell viability at the time of imaging was >90%. *, p<0.05 via ANOVA followed by Bonferroni post-test (N = 3±SEM).
Fig 5.
KIM-1 reporter cell line response to cisplatin and glucose.
Clones 5 and 9 demonstrate increased luciferase expression via IVIS imaging in response to increasing concentrations of cisplatin (A) or glucose (B). Cell viability at the time of imaging was >90%. *, p<0.05 via ANOVA followed by Bonferroni post-test (N = 3±SEM).
Fig 6.
Clone 5 retains the response to stimuli after deletion of the RFP-T2A-Puro cassette with Cre recombinase.
(A), schematic of cre-mediated deletion of RFP-T2A-Puro cassette at the genomic level. (B–D), RFP-T2A-Puro deleted cells response to hypoxia, cisplatin, and glucose respectively. Cell viability at the time of imaging was >90%. *, p<0.05 via ANOVA followed by Bonferroni post-test (N = 3±SEM).
Fig 7.
Lack of Cas9-mediated gene tagging of KIM-1 in RPTEC cells.
RPTEC cells were transfected with jetPRIME (A), FuGENE-6 (B), and TransfeX (C) with TransfeX proving to be the most efficient. Despite good transfection efficiency, only 4 puromycin resistant clones were isolated. None of these clones demonstrated verified gene tagging via PCR (D). HK-2 clone 5 (HK-2-5) was used as a positive control. (-), untransfected cells.