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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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Schematic of the perpetual serial integration of DNA segments into the IIS‐alphoid^tetO‐HAC by the iterative integration system (IIS). (A) The starting integration platform cassette on the human artificial chromosome (HAC). In the empty platform cassette, the SV40 enhancer plus ferritin (SFM) promoter drives the expression of the eGFP‐hph‐TK (GHT) marker. The cells express enhanced green fluorescence protein (eGFP; cells look green), and are hygromycin resistant (hph) and ganciclovir sensitive (TK). (B) Type I carrier vector bearing the first DNA segment of interest (DNA1) is integrated into the platform cassette of the HAC by Cre recombinase and ΦC31 integrase, which are themselves expressed from the plasmid A139. Recombination between a Type I carrier vector and a platform cassette by Cre recombinase and ΦC31 integrase leads to replacement of the GHT marker by the Puro‐mCherry‐FcyFur (PCF) marker and integration of the DNA of interest (DNA1) into the platform cassette. Cells with correct integration are selected for using puromycin and ganciclovir. (C) Structure of the platform cassette after the first round of DNA integration. The PCF marker is expressed. Therefore, the cells express red fluorescence (mCherry; cells now look red) and are puromycin (Puro) resistant and 5‐fluorocytosine (FcyFur) sensitive. (D) Recombination between a Type II carrier vector bearing the second DNA segment of interest (DNA2) and a platform cassette by Cre recombinase and ΦBT1 integrase, which are expressed from the plasmid A135‐JH, leads to replacement of the PCF marker by the GHT marker and DNA2 integration into the platform cassette. The integration event is selected for using hygromycin and 5‐fluorocytosine. (E) Structure of the platform cassette after the second round of DNA integration. The cells express the GHT marker and, thus, the green florescence protein eGFP (cells look green). They once again become hygromycin resistant (hph) and ganciclovir sensitive (TK). This structure is identical to the starting cassette aside from the integration of DNA segments of interest, DNA1 and DNA2.