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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

Efficient generation of Doxycyline-inducible stable cell lines free of background expression.(a) Knock out Maged1 Mouse Embryonic Fibroblasts (MEFs) were stably transduced with LVTPTIP-3xFlag-MAGED1b and treated with 1μg/ml Doxycycline for 4, 8 or 24hrs. Whole cell extracts were analysed for MAGED1b (anti-Flag) and tubulin by immunoblot. (b) LNCaP prostate cancer cells were stably transduced with LVTPTIP-SIM2s-3xFlag or LVTPTIP-SIM2l-3xFlag and treated with 1μg/ml Doxycycline for 4, 8 or 24hrs. Whole cell extracts were analysed for SIM2s or SIM2l (anti-Flag) and tubulin by immunoblot. (c) mRNA of SIM2s and SIM2l from LNCaP prostate cancer cells from (b) was measured by Real-time PCR and normalised to RNA polymerase subunit 2A (POLR2A). Data are mean normalised mRNA fold induction over parent cell line ± SEM of 3 independent experiments. Statistical significance was determined using an unpaired student t-test. n.s. (not significant).
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pone.0116373.g003: Efficient generation of Doxycyline-inducible stable cell lines free of background expression.(a) Knock out Maged1 Mouse Embryonic Fibroblasts (MEFs) were stably transduced with LVTPTIP-3xFlag-MAGED1b and treated with 1μg/ml Doxycycline for 4, 8 or 24hrs. Whole cell extracts were analysed for MAGED1b (anti-Flag) and tubulin by immunoblot. (b) LNCaP prostate cancer cells were stably transduced with LVTPTIP-SIM2s-3xFlag or LVTPTIP-SIM2l-3xFlag and treated with 1μg/ml Doxycycline for 4, 8 or 24hrs. Whole cell extracts were analysed for SIM2s or SIM2l (anti-Flag) and tubulin by immunoblot. (c) mRNA of SIM2s and SIM2l from LNCaP prostate cancer cells from (b) was measured by Real-time PCR and normalised to RNA polymerase subunit 2A (POLR2A). Data are mean normalised mRNA fold induction over parent cell line ± SEM of 3 independent experiments. Statistical significance was determined using an unpaired student t-test. n.s. (not significant).

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)

Efficient generation of Doxycyline-inducible stable cell lines free of background expression.(a) Knock out Maged1 Mouse Embryonic Fibroblasts (MEFs) were stably transduced with LVTPTIP-3xFlag-MAGED1b and treated with 1μg/ml Doxycycline for 4, 8 or 24hrs. Whole cell extracts were analysed for MAGED1b (anti-Flag) and tubulin by immunoblot. (b) LNCaP prostate cancer cells were stably transduced with LVTPTIP-SIM2s-3xFlag or LVTPTIP-SIM2l-3xFlag and treated with 1μg/ml Doxycycline for 4, 8 or 24hrs. Whole cell extracts were analysed for SIM2s or SIM2l (anti-Flag) and tubulin by immunoblot. (c) mRNA of SIM2s and SIM2l from LNCaP prostate cancer cells from (b) was measured by Real-time PCR and normalised to RNA polymerase subunit 2A (POLR2A). Data are mean normalised mRNA fold induction over parent cell line ± SEM of 3 independent experiments. Statistical significance was determined using an unpaired student t-test. n.s. (not significant).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0116373.g003: Efficient generation of Doxycyline-inducible stable cell lines free of background expression.(a) Knock out Maged1 Mouse Embryonic Fibroblasts (MEFs) were stably transduced with LVTPTIP-3xFlag-MAGED1b and treated with 1μg/ml Doxycycline for 4, 8 or 24hrs. Whole cell extracts were analysed for MAGED1b (anti-Flag) and tubulin by immunoblot. (b) LNCaP prostate cancer cells were stably transduced with LVTPTIP-SIM2s-3xFlag or LVTPTIP-SIM2l-3xFlag and treated with 1μg/ml Doxycycline for 4, 8 or 24hrs. Whole cell extracts were analysed for SIM2s or SIM2l (anti-Flag) and tubulin by immunoblot. (c) mRNA of SIM2s and SIM2l from LNCaP prostate cancer cells from (b) was measured by Real-time PCR and normalised to RNA polymerase subunit 2A (POLR2A). Data are mean normalised mRNA fold induction over parent cell line ± SEM of 3 independent experiments. Statistical significance was determined using an unpaired student t-test. n.s. (not significant).
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