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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

Lentiviral LVTPT system allows for inducible cDNA and shRNA expression in stable cell lines and primary neurons.(a) Vector diagram of constitutively expressed (Tet-On 3G and eGFP) and Doxycycline-inducible (dsRed (cDNA), Tet-On 3G and eGFP) components of the LVTPT system (b) dsRed (left), eGFP (middle), or merged (right) fluorescence images of HEK293T cells infected with LVTPTIG-dsRed virus in the absence (lower panel) or presence (upper panel) of 1μg/ml Doxycycline for 24hrs (c) FACS analysis of dsRed expressing cells in uninfected (orange) or HEK293T cells stably infected with LVTPTIP-dsRed-shGL3 and selected with Puromycin in the absence (red) or presence of 1μg/ml Doxycycline for 24 (green) or 48 (blue) hrs. (d) LVTPTIP elicits effective RNAi in HEK293T cells. HEK293T cells stably infected with LVTPTIP-dsRED-shGL3 (negative control), LVTPTIP-dsRED-shNPAS4 2182 (impotent shRNA), or LVTPTIP-dsRED-shNPAS4 275134 (potent shRNA) were transfected with expression vector pEFBOS-NPAS4–3xFlag and treated with (+) or without (-) 1 μg/ml Doxycycline for 72 hrs. Whole cell extracts were analysed for NPAS4 (anti-Flag) and tubulin levels by immunoblot. (e) dsRed fluorescence images of embryonic day 16 mouse cortical neurons cultured in vitro and infected with concentrated LVTPT-dsRed-shGL3 virus and treated with (right panel) or without (left panel) 1μg/ml Doxycycline for 24hrs.
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pone.0116373.g002: Lentiviral LVTPT system allows for inducible cDNA and shRNA expression in stable cell lines and primary neurons.(a) Vector diagram of constitutively expressed (Tet-On 3G and eGFP) and Doxycycline-inducible (dsRed (cDNA), Tet-On 3G and eGFP) components of the LVTPT system (b) dsRed (left), eGFP (middle), or merged (right) fluorescence images of HEK293T cells infected with LVTPTIG-dsRed virus in the absence (lower panel) or presence (upper panel) of 1μg/ml Doxycycline for 24hrs (c) FACS analysis of dsRed expressing cells in uninfected (orange) or HEK293T cells stably infected with LVTPTIP-dsRed-shGL3 and selected with Puromycin in the absence (red) or presence of 1μg/ml Doxycycline for 24 (green) or 48 (blue) hrs. (d) LVTPTIP elicits effective RNAi in HEK293T cells. HEK293T cells stably infected with LVTPTIP-dsRED-shGL3 (negative control), LVTPTIP-dsRED-shNPAS4 2182 (impotent shRNA), or LVTPTIP-dsRED-shNPAS4 275134 (potent shRNA) were transfected with expression vector pEFBOS-NPAS4–3xFlag and treated with (+) or without (-) 1 μg/ml Doxycycline for 72 hrs. Whole cell extracts were analysed for NPAS4 (anti-Flag) and tubulin levels by immunoblot. (e) dsRed fluorescence images of embryonic day 16 mouse cortical neurons cultured in vitro and infected with concentrated LVTPT-dsRed-shGL3 virus and treated with (right panel) or without (left panel) 1μg/ml Doxycycline for 24hrs.

Mentions: These lentiviral constructs were efficient in producing high virus titre and transducing a number of different mouse and human cell lines (Figs. 2, 3 and S1 Fig.). Initially, we tested these constructs in HEK293T cells stably transduced with a construct containing dsRed downstream of the TRE3G promoter (Fig. 2a). In the absence of Dox, dsRed was undetectable, while EGFP driven from the PGK promoter was expressed in transduced cells (Fig. 2b). After 24hrs of Dox treatment dsRed was robustly expressed throughout the EGFP-expressing population, indicating that the lentiviral constructs can produce complete populations of expressing cells (Fig. 2b). As previously reported, the Dox-induced expression also increased expression from the downstream PGK promoter as apparent by an increase in EGFP signal (Fig. 2b). This is presumably due to transcriptional read-through from the binding and activation of Tet-activator and the lack of polyadenylation sequences in lentivector constructs [23,24] (Fig. 2b). This allows for positive feed-forward expression of the Tet-On 3G, strengthening induction from the Tet-responsive TRE3G promoter.


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)

Lentiviral LVTPT system allows for inducible cDNA and shRNA expression in stable cell lines and primary neurons.(a) Vector diagram of constitutively expressed (Tet-On 3G and eGFP) and Doxycycline-inducible (dsRed (cDNA), Tet-On 3G and eGFP) components of the LVTPT system (b) dsRed (left), eGFP (middle), or merged (right) fluorescence images of HEK293T cells infected with LVTPTIG-dsRed virus in the absence (lower panel) or presence (upper panel) of 1μg/ml Doxycycline for 24hrs (c) FACS analysis of dsRed expressing cells in uninfected (orange) or HEK293T cells stably infected with LVTPTIP-dsRed-shGL3 and selected with Puromycin in the absence (red) or presence of 1μg/ml Doxycycline for 24 (green) or 48 (blue) hrs. (d) LVTPTIP elicits effective RNAi in HEK293T cells. HEK293T cells stably infected with LVTPTIP-dsRED-shGL3 (negative control), LVTPTIP-dsRED-shNPAS4 2182 (impotent shRNA), or LVTPTIP-dsRED-shNPAS4 275134 (potent shRNA) were transfected with expression vector pEFBOS-NPAS4–3xFlag and treated with (+) or without (-) 1 μg/ml Doxycycline for 72 hrs. Whole cell extracts were analysed for NPAS4 (anti-Flag) and tubulin levels by immunoblot. (e) dsRed fluorescence images of embryonic day 16 mouse cortical neurons cultured in vitro and infected with concentrated LVTPT-dsRed-shGL3 virus and treated with (right panel) or without (left panel) 1μg/ml Doxycycline for 24hrs.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4358958&req=5

pone.0116373.g002: Lentiviral LVTPT system allows for inducible cDNA and shRNA expression in stable cell lines and primary neurons.(a) Vector diagram of constitutively expressed (Tet-On 3G and eGFP) and Doxycycline-inducible (dsRed (cDNA), Tet-On 3G and eGFP) components of the LVTPT system (b) dsRed (left), eGFP (middle), or merged (right) fluorescence images of HEK293T cells infected with LVTPTIG-dsRed virus in the absence (lower panel) or presence (upper panel) of 1μg/ml Doxycycline for 24hrs (c) FACS analysis of dsRed expressing cells in uninfected (orange) or HEK293T cells stably infected with LVTPTIP-dsRed-shGL3 and selected with Puromycin in the absence (red) or presence of 1μg/ml Doxycycline for 24 (green) or 48 (blue) hrs. (d) LVTPTIP elicits effective RNAi in HEK293T cells. HEK293T cells stably infected with LVTPTIP-dsRED-shGL3 (negative control), LVTPTIP-dsRED-shNPAS4 2182 (impotent shRNA), or LVTPTIP-dsRED-shNPAS4 275134 (potent shRNA) were transfected with expression vector pEFBOS-NPAS4–3xFlag and treated with (+) or without (-) 1 μg/ml Doxycycline for 72 hrs. Whole cell extracts were analysed for NPAS4 (anti-Flag) and tubulin levels by immunoblot. (e) dsRed fluorescence images of embryonic day 16 mouse cortical neurons cultured in vitro and infected with concentrated LVTPT-dsRed-shGL3 virus and treated with (right panel) or without (left panel) 1μg/ml Doxycycline for 24hrs.
Mentions: These lentiviral constructs were efficient in producing high virus titre and transducing a number of different mouse and human cell lines (Figs. 2, 3 and S1 Fig.). Initially, we tested these constructs in HEK293T cells stably transduced with a construct containing dsRed downstream of the TRE3G promoter (Fig. 2a). In the absence of Dox, dsRed was undetectable, while EGFP driven from the PGK promoter was expressed in transduced cells (Fig. 2b). After 24hrs of Dox treatment dsRed was robustly expressed throughout the EGFP-expressing population, indicating that the lentiviral constructs can produce complete populations of expressing cells (Fig. 2b). As previously reported, the Dox-induced expression also increased expression from the downstream PGK promoter as apparent by an increase in EGFP signal (Fig. 2b). This is presumably due to transcriptional read-through from the binding and activation of Tet-activator and the lack of polyadenylation sequences in lentivector constructs [23,24] (Fig. 2b). This allows for positive feed-forward expression of the Tet-On 3G, strengthening induction from the Tet-responsive TRE3G promoter.

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