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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 prediction of shRNA sequences for knockdown of ARNT1 or ARNT2 in mES_FLP-Inducer cells.Induced shRNA knockdown of (a) ARNT1 or (b) ARNT2 in mES-FLP-Inducer-nucTomato-m30c-shRNA cells treated with 1μg/ml Dox for 48hrs (+) versus untreated (-). Western blotting was used to detect ARNT1, ARNT2, and tubulin using α-ARNT1, α-ARNT2, or α-tubulin antibodies, respectively.
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pone.0116373.g005: Efficient prediction of shRNA sequences for knockdown of ARNT1 or ARNT2 in mES_FLP-Inducer cells.Induced shRNA knockdown of (a) ARNT1 or (b) ARNT2 in mES-FLP-Inducer-nucTomato-m30c-shRNA cells treated with 1μg/ml Dox for 48hrs (+) versus untreated (-). Western blotting was used to detect ARNT1, ARNT2, and tubulin using α-ARNT1, α-ARNT2, or α-tubulin antibodies, respectively.

Mentions: We tested the ability of this shRNA selection algorithm to effectively predict efficient knockdown from singly integrated constructs using our mES FLP-Inducer system. We choose to target two genes, Arnt and Arnt2, which express bHLH/PAS transcription factors in embryonic stem cells [29] and can be readily detected by western immunoblot. We cloned the top 10 shRNA sequences predicted by our algorithm against either Arnt or Arnt2 using a multiplex cloning technique (see methods) and generated mES-FLP-Inducer cell lines for each construct. We then tested each shRNA for knockdown of ARNT or ARNT2 protein following 48 hrs of Dox treatment and observed ~60% (12/20) of shRNA constructs could effectively knockdown gene expression (Fig. 5a and 5b).


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 prediction of shRNA sequences for knockdown of ARNT1 or ARNT2 in mES_FLP-Inducer cells.Induced shRNA knockdown of (a) ARNT1 or (b) ARNT2 in mES-FLP-Inducer-nucTomato-m30c-shRNA cells treated with 1μg/ml Dox for 48hrs (+) versus untreated (-). Western blotting was used to detect ARNT1, ARNT2, and tubulin using α-ARNT1, α-ARNT2, or α-tubulin antibodies, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0116373.g005: Efficient prediction of shRNA sequences for knockdown of ARNT1 or ARNT2 in mES_FLP-Inducer cells.Induced shRNA knockdown of (a) ARNT1 or (b) ARNT2 in mES-FLP-Inducer-nucTomato-m30c-shRNA cells treated with 1μg/ml Dox for 48hrs (+) versus untreated (-). Western blotting was used to detect ARNT1, ARNT2, and tubulin using α-ARNT1, α-ARNT2, or α-tubulin antibodies, respectively.
Mentions: We tested the ability of this shRNA selection algorithm to effectively predict efficient knockdown from singly integrated constructs using our mES FLP-Inducer system. We choose to target two genes, Arnt and Arnt2, which express bHLH/PAS transcription factors in embryonic stem cells [29] and can be readily detected by western immunoblot. We cloned the top 10 shRNA sequences predicted by our algorithm against either Arnt or Arnt2 using a multiplex cloning technique (see methods) and generated mES-FLP-Inducer cell lines for each construct. We then tested each shRNA for knockdown of ARNT or ARNT2 protein following 48 hrs of Dox treatment and observed ~60% (12/20) of shRNA constructs could effectively knockdown gene expression (Fig. 5a and 5b).

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