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Inducible and reversible lentiviral and Recombination Mediated Cassette Exchange (RMCE) systems for controlling gene expression.

Bersten DC, Sullivan AE, Li D, Bhakti V, Bent SJ, Whitelaw ML - PLoS ONE (2015)

Bottom Line: We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs.We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs.These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Biomedical Science (Biochemistry), The University of Adelaide, Adelaide, South Australia, Australia; Institute of Molecular Pathology, The University of Adelaide, Adelaide, South Australia, Australia.

ABSTRACT
Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.

No MeSH data available.


Related in: MedlinePlus

Construction of sensitive, low background Dox-inducible lentiviral and Flp-In systems.(a) Tetracycline-responsive luciferase reporter gene assays in HEK293T cells using Tet-On variants with (black) or without (grey) 1μg/ml Doxycycline for 24hrs. (b) Expanded background luciferase activities in the absence of Doxycycline from (a). Data are mean relative luciferase activities ± SEM of 3 independent experiments. (c) The sensitivity of Tet-On or Tet-On 3G to Doxycycline using a Tetracycline-responsive luciferase reporter gene in HEK293T cells in the presence (black) or absence (grey) of indicated concentrations of Doxycycline. Data show fold inductions of mean relative luciferase for Doxycycline treated versus non-treated cells, ± SEM of 3 independent experiments. (d) Schematic of vectors for fluorescent miR30c donor plasmids containing dsRed, dnucTomato, or eYFP. (e) Schematic of vectors for Lentiviral (LVTPTIG and LVTPTIP) and FLP-Inducer (Col1a1-FLP-Inducer and Col1a1-FLP-Inducer-LSL) Doxycycline-inducible destination plasmids.
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pone.0116373.g001: Construction of sensitive, low background Dox-inducible lentiviral and Flp-In systems.(a) Tetracycline-responsive luciferase reporter gene assays in HEK293T cells using Tet-On variants with (black) or without (grey) 1μg/ml Doxycycline for 24hrs. (b) Expanded background luciferase activities in the absence of Doxycycline from (a). Data are mean relative luciferase activities ± SEM of 3 independent experiments. (c) The sensitivity of Tet-On or Tet-On 3G to Doxycycline using a Tetracycline-responsive luciferase reporter gene in HEK293T cells in the presence (black) or absence (grey) of indicated concentrations of Doxycycline. Data show fold inductions of mean relative luciferase for Doxycycline treated versus non-treated cells, ± SEM of 3 independent experiments. (d) Schematic of vectors for fluorescent miR30c donor plasmids containing dsRed, dnucTomato, or eYFP. (e) Schematic of vectors for Lentiviral (LVTPTIG and LVTPTIP) and FLP-Inducer (Col1a1-FLP-Inducer and Col1a1-FLP-Inducer-LSL) Doxycycline-inducible destination plasmids.

Mentions: Significant improvements have recently been made to the Tet-On inducible expression systems, including the molecular evolution of high sensitivity, low background variants of the Tet-activator and low background Tet- responsive promoters [3,5]. Before incorporating these components into lentiviral or RMCE expression systems, we confirmed these improvements to the Tet-activator using luciferase reporter assays containing a minimal CMV promoter and seven optimised Tet response element (TRE3G) repeats [5]. We observed strong Dox-inducible expression from all Tet-On variants (Fig. 1a), however significant background “leaky” expression was observed in the absence of Dox when either the wildtype (WT) Tet-On or the Tet-On V16 variants were employed as Tet-activators in HEK293T cells (Fig. 1b). Activation of the TRE promoter in the absence of Dox was negligible when the Tet-On 3G variant was used (Fig. 1b). The inability to gain Dox-inducible expression in some tissues, as described by several groups using the Tet-activator system, has been suggested to be due to low Dox concentration in these tissues. This issue may be overcome by using more sensitive Tet-activator variants [2,21,22]. We therefore tested the sensitivity of Tet-On 3G to Dox, and observed that the Tet-On 3G variant was active at Dox concentrations as low as 1ng/ml, revealing dramatically increased sensitivity to Dox compared to the Tet-On (Fig. 1c).


Inducible and reversible lentiviral and Recombination Mediated Cassette Exchange (RMCE) systems for controlling gene expression.

Bersten DC, Sullivan AE, Li D, Bhakti V, Bent SJ, Whitelaw ML - PLoS ONE (2015)

Construction of sensitive, low background Dox-inducible lentiviral and Flp-In systems.(a) Tetracycline-responsive luciferase reporter gene assays in HEK293T cells using Tet-On variants with (black) or without (grey) 1μg/ml Doxycycline for 24hrs. (b) Expanded background luciferase activities in the absence of Doxycycline from (a). Data are mean relative luciferase activities ± SEM of 3 independent experiments. (c) The sensitivity of Tet-On or Tet-On 3G to Doxycycline using a Tetracycline-responsive luciferase reporter gene in HEK293T cells in the presence (black) or absence (grey) of indicated concentrations of Doxycycline. Data show fold inductions of mean relative luciferase for Doxycycline treated versus non-treated cells, ± SEM of 3 independent experiments. (d) Schematic of vectors for fluorescent miR30c donor plasmids containing dsRed, dnucTomato, or eYFP. (e) Schematic of vectors for Lentiviral (LVTPTIG and LVTPTIP) and FLP-Inducer (Col1a1-FLP-Inducer and Col1a1-FLP-Inducer-LSL) Doxycycline-inducible destination plasmids.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358958&req=5

pone.0116373.g001: Construction of sensitive, low background Dox-inducible lentiviral and Flp-In systems.(a) Tetracycline-responsive luciferase reporter gene assays in HEK293T cells using Tet-On variants with (black) or without (grey) 1μg/ml Doxycycline for 24hrs. (b) Expanded background luciferase activities in the absence of Doxycycline from (a). Data are mean relative luciferase activities ± SEM of 3 independent experiments. (c) The sensitivity of Tet-On or Tet-On 3G to Doxycycline using a Tetracycline-responsive luciferase reporter gene in HEK293T cells in the presence (black) or absence (grey) of indicated concentrations of Doxycycline. Data show fold inductions of mean relative luciferase for Doxycycline treated versus non-treated cells, ± SEM of 3 independent experiments. (d) Schematic of vectors for fluorescent miR30c donor plasmids containing dsRed, dnucTomato, or eYFP. (e) Schematic of vectors for Lentiviral (LVTPTIG and LVTPTIP) and FLP-Inducer (Col1a1-FLP-Inducer and Col1a1-FLP-Inducer-LSL) Doxycycline-inducible destination plasmids.
Mentions: Significant improvements have recently been made to the Tet-On inducible expression systems, including the molecular evolution of high sensitivity, low background variants of the Tet-activator and low background Tet- responsive promoters [3,5]. Before incorporating these components into lentiviral or RMCE expression systems, we confirmed these improvements to the Tet-activator using luciferase reporter assays containing a minimal CMV promoter and seven optimised Tet response element (TRE3G) repeats [5]. We observed strong Dox-inducible expression from all Tet-On variants (Fig. 1a), however significant background “leaky” expression was observed in the absence of Dox when either the wildtype (WT) Tet-On or the Tet-On V16 variants were employed as Tet-activators in HEK293T cells (Fig. 1b). Activation of the TRE promoter in the absence of Dox was negligible when the Tet-On 3G variant was used (Fig. 1b). The inability to gain Dox-inducible expression in some tissues, as described by several groups using the Tet-activator system, has been suggested to be due to low Dox concentration in these tissues. This issue may be overcome by using more sensitive Tet-activator variants [2,21,22]. We therefore tested the sensitivity of Tet-On 3G to Dox, and observed that the Tet-On 3G variant was active at Dox concentrations as low as 1ng/ml, revealing dramatically increased sensitivity to Dox compared to the Tet-On (Fig. 1c).

Bottom Line: We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs.We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs.These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Biomedical Science (Biochemistry), The University of Adelaide, Adelaide, South Australia, Australia; Institute of Molecular Pathology, The University of Adelaide, Adelaide, South Australia, Australia.

ABSTRACT
Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.

No MeSH data available.


Related in: MedlinePlus