Journal: Cell Cycle
Article Title: Telomere dynamics in human pluripotent stem cells
doi: 10.1080/15384101.2023.2285551
Figure Lengend Snippet: BJ deficient for telomerase fail to be reprogrammed into iPSCs. a. Generation of TERT knock-out BJ fibroblasts by targeting the exon2 of TERT using the CRISPR/Cas9 technology. The sequences of BJ clones with successful TERT knockout are indicated. b. Generation of TERC knock-out BJ fibroblasts. Two sgRNAs flanking TERC were designed to delete TERC . Sequence of BJ clone with successful TERC deletion is indicated. Image of DNA electrophoresis gel showing the successful deletion of TERC is shown. c. Reprogramming of BJ fibroblasts with (WT) or without telomerase genes ( TERT −/− & TERC −/− ). Representative images of alkaline phosphatase (AP) staining showing iPS colonies are shown. Bright field images showing iPS colonies (yellow arrow) on feeders (top) and iPSCs established on feeder-free culture (bottom). d. Representative images of western blot analyzing NANOG, OCT4, TERT and TRF1 in different iPS clones at indicated passages by western blot. e. Protein level quantification. Bars represent mean values and error bars the standard deviation. One-way ANOVA was used for statistical analysis. n=number of independent clones.
Article Snippet: The primary antibodies used were anti-TRF1 (1:1000; BED5, Bio-Rad), anti-TERT (1:1000; ab320320, abcam), anti-NANOG (1:1000; Cell Signaling, 4903), anti-OCT4 (1:1000; Cell Signaling, 2750), anti-SOX2 (1:1000; Cell Signaling, 3579), and anti-β-Action (1:500; Santa Cruz 47,778).
Techniques: Knock-Out, CRISPR, Clone Assay, Sequencing, Nucleic Acid Electrophoresis, Staining, Western Blot, Standard Deviation
