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

Brough R, Papanastasiou AM, Porter AC - BMC Mol. Biol. (2007)

Bottom Line: Transgenes encoding luciferase, I-SceI endonuclease or Rad52 were then inserted by SSR at a LoxP site adjacent to the GFP gene resulting stringent tet-regulated transgene expression.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.This approach will be particularly useful for transgenes whose products are very active at low concentrations and/or for comparisons of multiple related transgenes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Gene Targeting Group, Department of Haematology and MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College, London, UK. Rachel.Brough@icr.ac.uk <Rachel.Brough@icr.ac.uk>

ABSTRACT

Background: 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.

Results: 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-SceI endonuclease or Rad52 were then inserted by SSR at a LoxP 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-SceI transgenes.

Conclusion: 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.

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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.
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Figure 1: 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.

Mentions: An outline of the basic Screen and Insert approach is shown in Fig. 1A. A target construct is made in which a reporter gene (EGFP) is expressed from a tet-responsive promoter (TRP = TRE + CMVmin) with a loxP site positioned between the TRP and the reporter gene. Stably transfected clones, each with the target construct randomly integrated at a different site, are screened by flow cytometry to identify those with stringently tet-regulated EGFP expression. A chosen clone is then co-transfected with a Cre-expression plasmid and an insertion construct in which a promoterless GOI cassette is linked to a loxP site. Cre-mediated recombination between the insertion construct and the integrated target construct places the GOI under the control of the TRP in the chosen clone. Correct insertion events generate clones with stringently tet-regulated GOI and no GFP expression.


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

Brough R, Papanastasiou AM, Porter AC - BMC Mol. Biol. (2007)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1884169&req=5

Figure 1: 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.
Mentions: An outline of the basic Screen and Insert approach is shown in Fig. 1A. A target construct is made in which a reporter gene (EGFP) is expressed from a tet-responsive promoter (TRP = TRE + CMVmin) with a loxP site positioned between the TRP and the reporter gene. Stably transfected clones, each with the target construct randomly integrated at a different site, are screened by flow cytometry to identify those with stringently tet-regulated EGFP expression. A chosen clone is then co-transfected with a Cre-expression plasmid and an insertion construct in which a promoterless GOI cassette is linked to a loxP site. Cre-mediated recombination between the insertion construct and the integrated target construct places the GOI under the control of the TRP in the chosen clone. Correct insertion events generate clones with stringently tet-regulated GOI and no GFP expression.

Bottom Line: Transgenes encoding luciferase, I-SceI endonuclease or Rad52 were then inserted by SSR at a LoxP site adjacent to the GFP gene resulting stringent tet-regulated transgene expression.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.This approach will be particularly useful for transgenes whose products are very active at low concentrations and/or for comparisons of multiple related transgenes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Gene Targeting Group, Department of Haematology and MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College, London, UK. Rachel.Brough@icr.ac.uk <Rachel.Brough@icr.ac.uk>

ABSTRACT

Background: 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.

Results: 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-SceI endonuclease or Rad52 were then inserted by SSR at a LoxP 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-SceI transgenes.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus