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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

Inducible, background free expression of cDNA from the Col1a1 locus(a) Col1a1-FLP-Inducer-mNPAS4–3xFlag mouse embryonic stem (mES) cells were cultured in the absence or presence of 1μg/ml Dox for 6 hrs and whole cell extracts analysed for NPAS4–3xFlag by immunoblotting with anti-Flag antibodies. n.s. represents a nonspecific band (b) Npas4 mRNA was measured using real time PCR of Col1a1-FLP parental cells, Col1a1-FLP-Inducer-mNPAS4–3xFlag cultured in the absence or presence of 1μg/ml Dox for 6hrs, and mouse embryonic day 16 (E16) cortical neurons cultured in vitro and pre-treated with 2μM Tetrodotoxin (TTX) and 100μM D (-)-2-amino-5-phosphopentanoic acid (AP-5) (TTX/AP5) channel blockers for 24hrs prior to 55mM KCl depolarisation treatment for 1hr. Data are mean ± SD of 3 independent experiments. Statistical analysis was calculated using two-tailed students t-test of 3 independent experiments. not significant (n.s.) (c) Bdnf mRNA was measured using real time PCR of Col1a1-FLP parental cells and Col1a1-FLP-Inducer-mNPAS4–3xFlag cultured in the absence or presence of 1μg/ml Dox for 6hrs (d) Col1a1-FLP parental cells (mES) or Col1a1-FLP-Inducer-mNPAS4–3xFlag cells were cultured in the absence or presence of indicated concentrations of Dox for 24 hrs and whole cell extracts analysed for NPAS4–3xFlag by immunoblotting with anti-Flag antibodies. n.s. represents a nonspecific band.
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pone.0116373.g006: Inducible, background free expression of cDNA from the Col1a1 locus(a) Col1a1-FLP-Inducer-mNPAS4–3xFlag mouse embryonic stem (mES) cells were cultured in the absence or presence of 1μg/ml Dox for 6 hrs and whole cell extracts analysed for NPAS4–3xFlag by immunoblotting with anti-Flag antibodies. n.s. represents a nonspecific band (b) Npas4 mRNA was measured using real time PCR of Col1a1-FLP parental cells, Col1a1-FLP-Inducer-mNPAS4–3xFlag cultured in the absence or presence of 1μg/ml Dox for 6hrs, and mouse embryonic day 16 (E16) cortical neurons cultured in vitro and pre-treated with 2μM Tetrodotoxin (TTX) and 100μM D (-)-2-amino-5-phosphopentanoic acid (AP-5) (TTX/AP5) channel blockers for 24hrs prior to 55mM KCl depolarisation treatment for 1hr. Data are mean ± SD of 3 independent experiments. Statistical analysis was calculated using two-tailed students t-test of 3 independent experiments. not significant (n.s.) (c) Bdnf mRNA was measured using real time PCR of Col1a1-FLP parental cells and Col1a1-FLP-Inducer-mNPAS4–3xFlag cultured in the absence or presence of 1μg/ml Dox for 6hrs (d) Col1a1-FLP parental cells (mES) or Col1a1-FLP-Inducer-mNPAS4–3xFlag cells were cultured in the absence or presence of indicated concentrations of Dox for 24 hrs and whole cell extracts analysed for NPAS4–3xFlag by immunoblotting with anti-Flag antibodies. n.s. represents a nonspecific band.

Mentions: Inducible expression of tagged proteins is of particular use in studying transcription factor occupancy of target sites in the genome and transcription factor function in protein complexes. However, excessive overexpression and issues with temporal control or background expression can lead to false positive discovery in such experiments. We therefore created FLP-Inducer cell lines containing Flag-tagged Npas4 to assess background and inducible expression levels of a prototypical cDNA in this system. We observed rapid induction of NPAS4–3xFlag protein expression following 6hrs of Dox addition in the mES cells (Fig. 6a). Npas4 mRNA and protein expression is highly restricted to the brain and upregulated following neuronal depolarisation [30,31]. To assess the level of expression compared to endogenously expressed Npas4 we compared mRNA of Col1a1-FLP-Inducer mES cells to mouse cortical neurons cultured in vitro and depolarised with 55mM KCl for 1hr. After 6hrs of Dox treatment Npas4 mRNA levels were similar to the endogenous levels in KCl depolarised neurons (Fig. 6b). Importantly, we also observed no statistical difference between expression of Npas4 in parental mES cells or untreated mES-FLP Inducer-NPAS4–3xFlag cells (Fig. 6b) indicating a distinct lack of background “leaky” expression compared to previous Tet-On systems expressing cDNAs from the Col1a1 locus [32,33]. Ectopic NPAS4 expression in mES-FLP-Inducer–NPAS4–3xFlag cells was also able to activate the downstream target gene Bdnf after 6hrs of Dox induction, indicating that short periods of Dox induction could potentially be used to identify primary target genes of transcription factors (Fig. 6C). Many tissues have poor permeability or delivery of Dox and are therefore exposed to lower Dox concentrations, thus strong activation of the Tet-On protein at low Dox concentrations is important for inducible expression in some tissues. Using the FLP-Inducer-NPAS4–3xFlag mES cells we found robust protein expression following 24hrs of treatment with Dox at concentrations as low as 10ng/ml (Fig. 6D).


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)

Inducible, background free expression of cDNA from the Col1a1 locus(a) Col1a1-FLP-Inducer-mNPAS4–3xFlag mouse embryonic stem (mES) cells were cultured in the absence or presence of 1μg/ml Dox for 6 hrs and whole cell extracts analysed for NPAS4–3xFlag by immunoblotting with anti-Flag antibodies. n.s. represents a nonspecific band (b) Npas4 mRNA was measured using real time PCR of Col1a1-FLP parental cells, Col1a1-FLP-Inducer-mNPAS4–3xFlag cultured in the absence or presence of 1μg/ml Dox for 6hrs, and mouse embryonic day 16 (E16) cortical neurons cultured in vitro and pre-treated with 2μM Tetrodotoxin (TTX) and 100μM D (-)-2-amino-5-phosphopentanoic acid (AP-5) (TTX/AP5) channel blockers for 24hrs prior to 55mM KCl depolarisation treatment for 1hr. Data are mean ± SD of 3 independent experiments. Statistical analysis was calculated using two-tailed students t-test of 3 independent experiments. not significant (n.s.) (c) Bdnf mRNA was measured using real time PCR of Col1a1-FLP parental cells and Col1a1-FLP-Inducer-mNPAS4–3xFlag cultured in the absence or presence of 1μg/ml Dox for 6hrs (d) Col1a1-FLP parental cells (mES) or Col1a1-FLP-Inducer-mNPAS4–3xFlag cells were cultured in the absence or presence of indicated concentrations of Dox for 24 hrs and whole cell extracts analysed for NPAS4–3xFlag by immunoblotting with anti-Flag antibodies. n.s. represents a nonspecific band.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4358958&req=5

pone.0116373.g006: Inducible, background free expression of cDNA from the Col1a1 locus(a) Col1a1-FLP-Inducer-mNPAS4–3xFlag mouse embryonic stem (mES) cells were cultured in the absence or presence of 1μg/ml Dox for 6 hrs and whole cell extracts analysed for NPAS4–3xFlag by immunoblotting with anti-Flag antibodies. n.s. represents a nonspecific band (b) Npas4 mRNA was measured using real time PCR of Col1a1-FLP parental cells, Col1a1-FLP-Inducer-mNPAS4–3xFlag cultured in the absence or presence of 1μg/ml Dox for 6hrs, and mouse embryonic day 16 (E16) cortical neurons cultured in vitro and pre-treated with 2μM Tetrodotoxin (TTX) and 100μM D (-)-2-amino-5-phosphopentanoic acid (AP-5) (TTX/AP5) channel blockers for 24hrs prior to 55mM KCl depolarisation treatment for 1hr. Data are mean ± SD of 3 independent experiments. Statistical analysis was calculated using two-tailed students t-test of 3 independent experiments. not significant (n.s.) (c) Bdnf mRNA was measured using real time PCR of Col1a1-FLP parental cells and Col1a1-FLP-Inducer-mNPAS4–3xFlag cultured in the absence or presence of 1μg/ml Dox for 6hrs (d) Col1a1-FLP parental cells (mES) or Col1a1-FLP-Inducer-mNPAS4–3xFlag cells were cultured in the absence or presence of indicated concentrations of Dox for 24 hrs and whole cell extracts analysed for NPAS4–3xFlag by immunoblotting with anti-Flag antibodies. n.s. represents a nonspecific band.
Mentions: Inducible expression of tagged proteins is of particular use in studying transcription factor occupancy of target sites in the genome and transcription factor function in protein complexes. However, excessive overexpression and issues with temporal control or background expression can lead to false positive discovery in such experiments. We therefore created FLP-Inducer cell lines containing Flag-tagged Npas4 to assess background and inducible expression levels of a prototypical cDNA in this system. We observed rapid induction of NPAS4–3xFlag protein expression following 6hrs of Dox addition in the mES cells (Fig. 6a). Npas4 mRNA and protein expression is highly restricted to the brain and upregulated following neuronal depolarisation [30,31]. To assess the level of expression compared to endogenously expressed Npas4 we compared mRNA of Col1a1-FLP-Inducer mES cells to mouse cortical neurons cultured in vitro and depolarised with 55mM KCl for 1hr. After 6hrs of Dox treatment Npas4 mRNA levels were similar to the endogenous levels in KCl depolarised neurons (Fig. 6b). Importantly, we also observed no statistical difference between expression of Npas4 in parental mES cells or untreated mES-FLP Inducer-NPAS4–3xFlag cells (Fig. 6b) indicating a distinct lack of background “leaky” expression compared to previous Tet-On systems expressing cDNAs from the Col1a1 locus [32,33]. Ectopic NPAS4 expression in mES-FLP-Inducer–NPAS4–3xFlag cells was also able to activate the downstream target gene Bdnf after 6hrs of Dox induction, indicating that short periods of Dox induction could potentially be used to identify primary target genes of transcription factors (Fig. 6C). Many tissues have poor permeability or delivery of Dox and are therefore exposed to lower Dox concentrations, thus strong activation of the Tet-On protein at low Dox concentrations is important for inducible expression in some tissues. Using the FLP-Inducer-NPAS4–3xFlag mES cells we found robust protein expression following 24hrs of treatment with Dox at concentrations as low as 10ng/ml (Fig. 6D).

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