Journal: ACS Synthetic Biology
Article Title: Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies
Figure Lengend Snippet: Hybrid HAC formation in HT1080 cells. (a) Representative FISH images of clones containing a HAC (left) and an array integration in an endogenous chromosome (right). (b, c) Screening of blasticidin-resistant clones by FISH. Diagrams represent the frequency of metaphases with HACs (black bars) and array integrations (gray bars) ( N = 25) in HT1080 cells without (b) and with (c) CENP-A overexpression. (d) Frequency of HAC-containing clones with (CENP-A OE) and without (CENP-A WT) transient CENP-A overexpression during HAC formation. Only clones with a minimum of 10% metaphases containing HACs were considered as positive (10 vs 33%). (e) Representative two-color oligo-FISH images showing different hybrid HACs (clone 20.CA.07-top and 20.CA.24-bottom) containing tetO (red) and lacOgal4 (green) domains. Images were captured at optimized exposure times to clearly distinguish both signals in either clone (for signal intensity comparison between clones, see Figure S2 ). (f) Representative image of an HT1080 cell containing HAC clone 20.CA.24 and expressing both lacI-GFP (green) and tetR-mCherry (red) fusion proteins. Merged image (right panel) represents the overlay of GFP, mCherry, and DAPI channels. (g) Frequency of HAC-containing metaphases in the indicated clones containing HACs in the presence of blasticidin and after 30 days after blasticidin washout. The HAC loss rate is indicated in red. (h) Representative immunofluorescence images on metaphase spreads of HAC clone 20.CA.24 and stained with the indicated antibodies. Scale bars = 10 μm.
Article Snippet: For oligo-FISH experiments, oligonucleotides recognizing the tetO sequence (5′- ACTAGCAGCAGAGCTCTCCCTATCAGTGATAGAGACTAG-3′) labeled with Digoxigenin, and oligonucleotides recognizing both lacO (5′- CATGTGGAATTGTGAGCGGATAACAATTTGTGG-3′) and Gal4 (5′- TCGACGGAGGACAGTCCTCCG-3′) sequences labeled with Biotin were synthesized (Sigma).
Techniques: HAC Assay, Fluorescence In Situ Hybridization, Clone Assay, Over Expression, Expressing, Immunofluorescence, Staining