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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 and reversible expression and knockdown from the Col1a1 locus using the FLP-Inducer system.(a) Schematic representation of Col1a1-FLP-In targeting strategy. Electroporation of pFLP-Inducer-dsRed-shGL3 and pFLPo recombinase into mouse embryonic stem (mES) cells pre-targeted with Col1a1 FRT Hygromycin (–ATG) (promoter) enables gain of Hygromycin resistance and site specific integration of the FLP-Inducer construct. (b) Southern Blot of two mouse embryonic stem cell lines containing Col1a1-PGK-FRT-neo-FRT (–ATG) Hygro targeted to the Col1a1 locus and two G418 resistant WT lines, using a 5’ internal probe. (c) dsRed microscopy of a Col1a1-FLP-Inducer- dsRed-shGL3 Hygromycin-resistant mES cell line in the presence (right panel) or absence (left panel) of 1μg/ml Doxycycline for 24hrs. (d) FACS analysis of parent mES Col1a1 FRT Hygromycin (–ATG) (promoterless) cells (Orange) or mES Col1a1-FLP-Inducer- dsRed-shGL3 cells (blue and red) in the presence (red) or absence (blue and orange) of 1μg/ml Doxycycline for 24hrs. (e) Median dsRed fluorescence in FACS analysis in (d). (f) shRNA mediated knockdown of P53 from the Col1a1-FLP-Inducer mES cells. Col1a1-FLP-Inducer-dsRed-shGL3 or Col1a1-FLP-Inducer-dsRed-shP53 were incorporated into the Col1a1 locus in mES cells by recombination mediated cassette exchange (RMCE) and shRNA induced using 1μg/ml Doxycycline, then allowed to recover by Doxycycline removal for indicated periods. P53 and tubulin protein expression were determined by immunoblot.
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pone.0116373.g004: Inducible and reversible expression and knockdown from the Col1a1 locus using the FLP-Inducer system.(a) Schematic representation of Col1a1-FLP-In targeting strategy. Electroporation of pFLP-Inducer-dsRed-shGL3 and pFLPo recombinase into mouse embryonic stem (mES) cells pre-targeted with Col1a1 FRT Hygromycin (–ATG) (promoter) enables gain of Hygromycin resistance and site specific integration of the FLP-Inducer construct. (b) Southern Blot of two mouse embryonic stem cell lines containing Col1a1-PGK-FRT-neo-FRT (–ATG) Hygro targeted to the Col1a1 locus and two G418 resistant WT lines, using a 5’ internal probe. (c) dsRed microscopy of a Col1a1-FLP-Inducer- dsRed-shGL3 Hygromycin-resistant mES cell line in the presence (right panel) or absence (left panel) of 1μg/ml Doxycycline for 24hrs. (d) FACS analysis of parent mES Col1a1 FRT Hygromycin (–ATG) (promoterless) cells (Orange) or mES Col1a1-FLP-Inducer- dsRed-shGL3 cells (blue and red) in the presence (red) or absence (blue and orange) of 1μg/ml Doxycycline for 24hrs. (e) Median dsRed fluorescence in FACS analysis in (d). (f) shRNA mediated knockdown of P53 from the Col1a1-FLP-Inducer mES cells. Col1a1-FLP-Inducer-dsRed-shGL3 or Col1a1-FLP-Inducer-dsRed-shP53 were incorporated into the Col1a1 locus in mES cells by recombination mediated cassette exchange (RMCE) and shRNA induced using 1μg/ml Doxycycline, then allowed to recover by Doxycycline removal for indicated periods. P53 and tubulin protein expression were determined by immunoblot.

Mentions: In addition to lentiviral-mediated gene delivery to primary neurons and generation of stable cell lines, we were also interested in applying this technology to generate inducible expression from a defined locus in mouse embryonic stem (mES) cells. These modified mES cells can be used to study function of genes during in vitro differentiation protocols and in generating mouse models with inducible gain and loss of gene function. Following methods employed by the Jaenisch lab [11] we used a region 3’ of the coding sequence in the Col1a1 locus for targeted homologous recombination to insert a FLP/FRT based RMCE (Recombination Mediated Cassette Exchange) construct into hybrid S129/C57BL6 mES cells. RMCE allows rapid and site-specific exchange of single Dox-inducible cassettes into the Col1a1 locus and has been used to generate transgenic animals with ubiquitous Dox-inducible expression [11,27]. The Col1a1 targeting construct contained a Neomycin selection cassette flanked by FRT sites, followed by a Hygromycin cassette lacking a promoter or start codon as used by Jaenisch and colleagues [11,27] (Fig. 4a). We generated two independently derived mES cell lines by homologous recombination (F1 and F12 mES cell lines) and screened for correctly targeted single integrants by southern blot (Fig. 4a and 4b).


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 and reversible expression and knockdown from the Col1a1 locus using the FLP-Inducer system.(a) Schematic representation of Col1a1-FLP-In targeting strategy. Electroporation of pFLP-Inducer-dsRed-shGL3 and pFLPo recombinase into mouse embryonic stem (mES) cells pre-targeted with Col1a1 FRT Hygromycin (–ATG) (promoter) enables gain of Hygromycin resistance and site specific integration of the FLP-Inducer construct. (b) Southern Blot of two mouse embryonic stem cell lines containing Col1a1-PGK-FRT-neo-FRT (–ATG) Hygro targeted to the Col1a1 locus and two G418 resistant WT lines, using a 5’ internal probe. (c) dsRed microscopy of a Col1a1-FLP-Inducer- dsRed-shGL3 Hygromycin-resistant mES cell line in the presence (right panel) or absence (left panel) of 1μg/ml Doxycycline for 24hrs. (d) FACS analysis of parent mES Col1a1 FRT Hygromycin (–ATG) (promoterless) cells (Orange) or mES Col1a1-FLP-Inducer- dsRed-shGL3 cells (blue and red) in the presence (red) or absence (blue and orange) of 1μg/ml Doxycycline for 24hrs. (e) Median dsRed fluorescence in FACS analysis in (d). (f) shRNA mediated knockdown of P53 from the Col1a1-FLP-Inducer mES cells. Col1a1-FLP-Inducer-dsRed-shGL3 or Col1a1-FLP-Inducer-dsRed-shP53 were incorporated into the Col1a1 locus in mES cells by recombination mediated cassette exchange (RMCE) and shRNA induced using 1μg/ml Doxycycline, then allowed to recover by Doxycycline removal for indicated periods. P53 and tubulin protein expression were determined by immunoblot.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4358958&req=5

pone.0116373.g004: Inducible and reversible expression and knockdown from the Col1a1 locus using the FLP-Inducer system.(a) Schematic representation of Col1a1-FLP-In targeting strategy. Electroporation of pFLP-Inducer-dsRed-shGL3 and pFLPo recombinase into mouse embryonic stem (mES) cells pre-targeted with Col1a1 FRT Hygromycin (–ATG) (promoter) enables gain of Hygromycin resistance and site specific integration of the FLP-Inducer construct. (b) Southern Blot of two mouse embryonic stem cell lines containing Col1a1-PGK-FRT-neo-FRT (–ATG) Hygro targeted to the Col1a1 locus and two G418 resistant WT lines, using a 5’ internal probe. (c) dsRed microscopy of a Col1a1-FLP-Inducer- dsRed-shGL3 Hygromycin-resistant mES cell line in the presence (right panel) or absence (left panel) of 1μg/ml Doxycycline for 24hrs. (d) FACS analysis of parent mES Col1a1 FRT Hygromycin (–ATG) (promoterless) cells (Orange) or mES Col1a1-FLP-Inducer- dsRed-shGL3 cells (blue and red) in the presence (red) or absence (blue and orange) of 1μg/ml Doxycycline for 24hrs. (e) Median dsRed fluorescence in FACS analysis in (d). (f) shRNA mediated knockdown of P53 from the Col1a1-FLP-Inducer mES cells. Col1a1-FLP-Inducer-dsRed-shGL3 or Col1a1-FLP-Inducer-dsRed-shP53 were incorporated into the Col1a1 locus in mES cells by recombination mediated cassette exchange (RMCE) and shRNA induced using 1μg/ml Doxycycline, then allowed to recover by Doxycycline removal for indicated periods. P53 and tubulin protein expression were determined by immunoblot.
Mentions: In addition to lentiviral-mediated gene delivery to primary neurons and generation of stable cell lines, we were also interested in applying this technology to generate inducible expression from a defined locus in mouse embryonic stem (mES) cells. These modified mES cells can be used to study function of genes during in vitro differentiation protocols and in generating mouse models with inducible gain and loss of gene function. Following methods employed by the Jaenisch lab [11] we used a region 3’ of the coding sequence in the Col1a1 locus for targeted homologous recombination to insert a FLP/FRT based RMCE (Recombination Mediated Cassette Exchange) construct into hybrid S129/C57BL6 mES cells. RMCE allows rapid and site-specific exchange of single Dox-inducible cassettes into the Col1a1 locus and has been used to generate transgenic animals with ubiquitous Dox-inducible expression [11,27]. The Col1a1 targeting construct contained a Neomycin selection cassette flanked by FRT sites, followed by a Hygromycin cassette lacking a promoter or start codon as used by Jaenisch and colleagues [11,27] (Fig. 4a). We generated two independently derived mES cell lines by homologous recombination (F1 and F12 mES cell lines) and screened for correctly targeted single integrants by southern blot (Fig. 4a and 4b).

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