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Fig. 3 | Journal of Hematology & Oncology

Fig. 3

From: PTENP1 is a ceRNA for PTEN: it’s CRISPR clear

Fig. 3

CRISPR-mediated knock-in of a GFP expression cassette in PTENP1 gene results in the downregulation of PTEN expression and in an increase in DU145 cell proliferation. a Schematic representation of the CRISPR/Cas9-mediated cleavage of PTENP1 gene region, followed by the homology recombination-mediated knock-in of a GFP expression cassette. Being oriented in the opposite direction, the cassette interferes with the transcription of PTENP1 itself. The double-strand cut of genomic DNA produced by Cas9/sgRNA-PTENP1 (blue) is located within the 5′UTR region of PTENP1, upstream of the promoter of PTENP1 AS α and β. Therefore, the transcriptional unit of the antisense transcripts is preserved. Besides the arms required for homology recombination and the GFP expression cassette, the knock-in construct contains 2 insulators and 2 loxP sites. b Cartoon summarizing the experimental protocol used. DU145 that constitutively express Cas9 and sgRNA-PTENP1 were electroporated with pHR410PA-1-PTENP1 plasmid that contains the knock-in construct. Then, Cas9-mediated cleavage of PTENP1 genomic DNA was induced by adding doxycycline. After waiting 10–14 days in order to allow the dilution of non-integrated plasmid, cells that stably express GFP were sorted and seeded at 1-cell/96-well density to obtain individual knock-in clones. c PCR-based screening of the clones that are correctly knocked in. The junction regions located upstream and downstream of the knock-in construct were amplified using the primers shown in panel a as purple and dark red arrows, respectively. The purple forward and the dark red reverse primers (they give a PCR band only on wt alleles, not on knocked-in alleles) were also used to test whether the integration of the construct occurred on all chromosome 9 copies or not (het). The genomic DNA extracted from parental DU145 cells was used as negative control (C−). The genomic DNA extracted from DU145-GFP-KI cells right before 1-cell/96-well seeding was used as positive control (C+). Clones #A, 2, 5, 8, and 13 all show the correct integration of the construct, although non-knocked-in copies of chromosome 9 remain. d qRT-PCR quantification of the indicated transcripts in DU145 cells (taken as control) and clones #A, 2, 5, 8, and 13. e (left) Quantification of PTEN/GAPDH and pAKT/GAPDH protein levels in DU145 cells (taken as control) and in clones #A, 2, 5, 8, and 13. (right) Representative western blot detection of PTEN, pAKT and GAPDH proteins in DU145 cells and in clone #13. f Growth curves of DU145 cells and clones #A, 2, and 13. T1/3/5/7: days after seeding. g qRT-PCR quantification of the indicated transcripts 24 h after the electroporation of 1.5 μg of pGLU empty plasmid or of pGLU/ψ3′UTR plasmid in clone #13. The results reported in dg confirm what obtained by RNA interference and CRISPR/CasRx: the knock-down of PTENP1 negatively affects PTEN expression. As a consequence, AKT gets hyper-phosphorylated and cell proliferation increases. The graphs represent the mean ± SEM of three independent experiments. Statistically significant differences are indicated with asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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