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. 2021 Dec 17;1(12):e316. doi: 10.1002/cpz1.316

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Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC.

This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

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Generation of the alphoid^tetO‐HAC using a synthetic alphoid DNA array. (A) A 343‐bp synthetic alphoid dimer consists of two monomers. One monomer is derived from a chromosome 17 alphoid type I 16‐mer unit and contains a CENP‐B box, a nucleotide motif involved in centromere formation. The second monomer is a wholly synthetic sequence derived from alphoid DNA consensus with the sequence corresponding to the CENP‐B box replaced by a 42‐bp tetO motif. A dimer is amplified up to ∼3‐5 kb in size by rolling circle amplification (RCA) in vitro using phi29 DNA polymerase. Then the RCA‐amplified fragments are assembled by transformation‐associated recombination (TAR) cloning in yeast, leading to formation of an ∼50 kb synthetic array cloned into a YAC/BAC vector. A hybrid circular YAC/BAC vector contains a blasticidin resistance marker (a bsr gene). The YAC/BAC molecules are then moved from yeast to bacterial cells for further BAC DNA isolation. After transfection of 50 kb input BAC DNA into human HT1080 cells, the alphoid^tetO‐HAC is formed. Formation of the alphoid^tetO‐HAC is accompanied by multimerization of input 50 kb DNA up to 1.1 Mb. (B) FISH analysis of the alphoid^tetO‐HAC in human HT1080 cells. FISH analysis was performed using a fluorescein peptide nucleic acid (PNA)‐labeled probe for the tetO sequence (see Support Protocol). A white arrow indicates the HAC (green), while the endogenous chromosomes are labeled blue (DAPI). (C) Loss of the alphoid^tetO‐HAC from recipient cells may be induced by the transcriptional activator (tTA) fused with the tet‐repressor (tetR) targeting the tetO‐HAC kinetochore (Kim et al., 2011; Kononenko et al., 2014). (D) The IIS‐alphoid^tetO‐HAC was developed after insertion of the integration platform cassette into the alphoid^tetO‐HAC. The integration platform cassette consists of the SFM promoter driving the expression of the GHT marker, a loxP site present between the promoter and the marker, and the attB^ΦC31 site for the ΦC31 integrase. The GHT marker is a fusion of enhanced green fluorescent protein (eGFP), hygromycin‐B‐phosphotransferase (hph), and thymidine kinase (TK). Abbreviations: BAC, bacterial artificial chromosome; YAC, yeast artificial chromosome; HAC, human artificial chromosome; FISH, fluorescence in situ hybridization; SFM, SV40 enhancer plus ferritin; GHT, eGFP‐hph‐TK.