Mutant GTF2I induces cell transformation and metabolic alterations in thymic epithelial cells

摘要

a Strategy to knockin Gtf2i mutation in TEC71 using CRISPR/Cas9n. The Gtf2i-specific guide RNA (pU6-gRNA) sequences are in green, and the protospacer-adjacent motif (PAM) sequence is shown in red. The targeted allele is replaced with double-strand DNA template donor containing SphI restriction enzyme site (bold italic) and two point mutations (red). A red arrowhead indicates the mutation site of Gtf2i. PCR with outward primers (#1) and inward primers (#2) sequentially amplified the genomic region of Gtf2i to verify KI. b Point mutations (CTG to CAT) of Gtf2i identified by Sanger sequencing. TEC71 cells transfected with Cas9n and one gRNA were used as control. c DNA fragmentation by SphI enzyme was used to verify the correct replacement of Gtf2i gene with mutation. One microgram of PCR amplified DNA with primer set #2 was incubated with SphI for 3 h and DNA fragments (317 and 245 bp) were analyzed using 2% agarose gel electrophoresis. d Cytoskeleton and cell nuclei were stained with Texas Red-X phalloidin and DAPI, respectively, to enable observation of morphological changes. Arrows indicate cellular shape changes associated with cancer: irregular nuclear contour with polylobulation (yellow) or scant cytoplasm (white). Scale bars, 200 μm. e DNA-ploidy flow cytometric analysis of KI cells. f In vivo xenograft experiment of KI cells and control cells. Tumor volume (P < 0.0001) is presented as the means ± s.d. (n = 10 mice in each group). Statistical significance was determined by two-way ANOVA.