Journal: Molecular and Cellular Biology
Article Title: Ki-67 Contributes to Normal Cell Cycle Progression and Inactive X Heterochromatin in p21 Checkpoint-Proficient Human Cells
Figure Lengend Snippet: Validation of specificity and effectiveness of Ki-67 depletion reagents. (A) CRISPR/Cas9-based strategy for generating siRNA-resistant mutations in the endogenous Ki-67 gene. The si-Ki-67 target (green letters) and sgRNA-directed cleavage site (triangle) are indicated in the upper diagram of the endogenous locus. Altered nucleotides (red) and the novel EcoO109I restriction site are shown in the lower diagram of the repair template. (B) DNA sequence analysis of PCR products from wild-type hTERT-RPE1 cells and si-Ki-67-resistant clone 7. (C) EcoO109I digestion of the same PCR products sequenced for panel B. (D) Immunoblot analysis of clone 7 treated with the indicated reagents. (E) RT-qPCR analysis of clone 7. (F to L) Immunoblot analyses of the indicated cell lines treated with the indicated RNA depletion reagents.
Article Snippet: PCR products (1,523 bp) were further digested with the EcoO109I restriction enzyme (New England BioLabs).
Techniques: CRISPR, Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR