Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics

<p>Abstract</p> <p>Background</p> <p>The ability to regulate transgene expression has many applications, mostly concerning the analysis of gene function. Desirable induction characteristics, such as low un-induced expression, high induced expression and limited cellular heterogeneity, can be seriously impaired by chromosomal position effects at the site of transgene integration. Many clones may therefore need to be screened before one with optimal induction characteristics is identified. Furthermore, such screens must be repeated for each new transgene investigated, and comparisons between clones with different transgenes is complicated by their different integration sites.</p> <p>Results</p> <p>To circumvent these problems we have developed a "screen and insert" strategy in which clones carrying a transgene for a fluorescent reporter are first screened for those with optimal induction characteristics. Site-specific recombination (SSR) is then be used repeatedly to insert any new transgene at the reporter transgene locus of such clones so that optimal induction characteristics are conferred upon it. Here we have tested in a human fibrosarcoma cell line (HT1080) two of many possible implementations of this approach. Clones (e.g. Rht14-10) in which a GFP reporter gene is very stringently regulated by the tetracycline (tet) transactivator (tTA) protein were first identified flow-cytometrically. Transgenes encoding luciferase, I-<it>Sce</it>I endonuclease or Rad52 were then inserted by SSR at a <it>LoxP </it>site adjacent to the GFP gene resulting stringent tet-regulated transgene expression. In clone Rht14-10, increases in expression from essentially background levels (+tet) to more than 10⁴-fold above background (-tet) were reproducibly detected after Cre-mediated insertion of either the luciferase or the I-<it>Sce</it>I transgenes.</p> <p>Conclusion</p> <p>Although previous methods have made use of SSR to integrate transgenes at defined sites, none has effectively combined this with a pre-selection step to identify integration sites that support optimal regulatory characteristics. Rht14-10 and similar HT1080-derived clones can now be used in conjunction with a convenient delivery vector (pIN2-neoMCS), in a simple 3-step protocol leading to stringent and reproducible transgene regulation. This approach will be particularly useful for transgenes whose products are very active at low concentrations and/or for comparisons of multiple related transgenes.</p

Screen and Insert principles and strategies

Screen and Insert principles and strategies. Configurations of plasmid DNA before and after its incorporation into genomic DNA are shown schematically (not to scale). A) Principles of the Screen and Insert approach. B) The Screen and Insert type 1 (ScIn-1) strategy. C) The Screen and Insert type 2 (ScIn-2) strategy. Promoterless cassettes are shown for a generic gene of interest (GOI), and for genes encoding green fluorescent proteins (EGFP and d2EGFP), luciferase (luc), hygromycin phophotransferase (hygro) and guanosine phospribosyltranferase (gpt). See text for details. <p> </p> <p><b>Copyright information:</b></p><p>Taken from "Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics"</p><p>BMC Molecular Biology 2007;8():30-30.</p><p>Published online 10 May 2007</p><p>PMCID:PMC1884169.</p

Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics-1

<p><b>Copyright information:</b></p><p>Taken from "Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics"</p><p>BMC Molecular Biology 2007;8():30-30.</p><p>Published online 10 May 2007</p><p>PMCID:PMC1884169.</p><p></p>Flow cytometric profiles (y-axis, counts; x-axis, GFP fluorescence [FLH1]) of clones isolated after transfection with plasmids shown in A. For profiles in B (left only), C, D, F and G the green and yellow traces represent cells grown with or without tet, respectively, and the red traces represent GFP-negative cells (Rht14 or HT1080). . Left: Clone 6, the most stringently regulated clone isolated after transfection of pTARG1 into HT2 cells. Right: GFP expression profile of Clone 6 at indicated times after addition of tet. . Four of the most stringently regulated clones isolated after transfection of pTARG2 into HT2 cells. . Three of the most stringently regulated clones isolated after transfection of pTARG3 into HT2 cells. . Effect of AZC on GFP expression in T15, a pTARG3-transfected HT2 clone. Cells were cultured with (green) or without (blue) AZC for 24 h, and then for a further 24 h without AZC, before analysis. . Four of the most stringently regulated clones isolated after transfection of pTARG4 into Rht14 cells. . GFP expression profile of two heterogeneously expressing clones isolated after transfection of Rht14 with pTARG4. . Profile of clone Rht14-10 grown continuously with passaging for 14 d or 42 d. . GFP expression in clone Rht14-19, as measured by immunoblot (top) or flow cytometry (bottom), at the indicated times after addition of tet

Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics-3

<p><b>Copyright information:</b></p><p>Taken from "Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics"</p><p>BMC Molecular Biology 2007;8():30-30.</p><p>Published online 10 May 2007</p><p>PMCID:PMC1884169.</p><p></p>c). DNA is represented as in Fig. 1 with recognition sites for III () and II () and PCR primers indicated. . Ethidium bromide stained agarose gel of electrophoresed PCR products generated with the primers O1 and O3 and cell pellets of 9 MPA/X, GFP-negative clones selected after transfection of Rht14-10 (Rht14-10IN) or of Rht14-19 (Rht14-19IN) with pIN-2 and pMC-Cre. M = marker DNA. Negative (-) and positive (+) controls used, respectively, no DNA and pTIGHTgpt DNA (expected product: 555 bp). . Southern blots of genomic DNA isolated from Rht14-10 and derivatives and digested with II (left), or from Rht14-19 and derivatives and digested with III (right). Derivatives before (10IN5 and 19IN3, 4 and 5) and after (10flp9.2 and 3 and 19flp9.2 and 12.3) Flp-mediated deletion are analysed. The probe was a fragment of the gene (methods)

Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics-5

<p><b>Copyright information:</b></p><p>Taken from "Stringent and reproducible tetracycline-regulated transgene expression by site-specific insertion at chromosomal loci with pre-characterised induction characteristics"</p><p>BMC Molecular Biology 2007;8():30-30.</p><p>Published online 10 May 2007</p><p>PMCID:PMC1884169.</p><p></p>-mediated insertion of pIN2-neoSCE or pIN2-neoR52. . Ethidium bromide stained agarose gel of electrophoresed PCR products generated with the primers O2 and O5 on cell pellets of three pIN2-neoSCE- and three pIN2-neoR52-transfected Rht14-10 derivatives. Negative (-) and positive (+) controls were, respectively, no cells and cells (TetNeo, see methods) with an integrated tet-regulated neo cassette (expected product: 944 bp). Clones chosen for flp-mediated deletion (10IN-SCE.1 and 10IN-R52.1) are arrowed. . As in B but after Flp mediated deletion. . Ethidium bromide stained agarose gel of electrophoresed PCR products generated with the primers O2 and O6 on clones derived from 10IN-SCE.1 (1–12, left) and primers O2 and O7 on clones derived from 10IN-R52.1 (1–12, right). Control reaction contained no cells (-) or genomic DNA generated from pools of flp-treated pIN2-neoSCE/pIN2-neoR52 cells (+). Clones chosen for further analysis (10IN-SCE.1flp1 and 10IN-R52.1flp1) are arrowed. . Immunoblot analysis of I-I and Rad52 expression in Rht14-10 and, respectively in 10IN-SCE.1flp1 or 10IN-R52.1flp1 at the indicated times after removal or tetracycline