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Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells.

Kacherovsky N, Harkey MA, Blau CA, Giachelli CM, Pun SH - Nucleic Acids Res. (2012)

Bottom Line: The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern.Copy number analysis by quantitative PCR (qPCR) revealed that CID-selected cells contain on average higher copy numbers of transgenes than flow cytometry-selected cells, demonstrating selective advantage for cells with multiple transposon insertions.This non-viral, positive selection approach is an attractive alternative for producing engineered cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture times. Here, we introduce an efficient non-viral approach combining the Sleeping Beauty (SB) Transposon System with selective proliferation of engineered cells by chemically induced dimerization (CID) of growth factor receptors. Minicircles carrying a SB transposon cassette containing a reporter transgene and a gene for the F36VFGFR1 fusion protein were delivered to the hematopoietic cell line Ba/F3. Stably-transduced Ba/F3 cell populations with >98% purity were obtained within 1 week using this positive selection strategy. Copy number analysis by quantitative PCR (qPCR) revealed that CID-selected cells contain on average higher copy numbers of transgenes than flow cytometry-selected cells, demonstrating selective advantage for cells with multiple transposon insertions. A diverse population of cells is present both before and after culture in CID media, although site-specific qPCR of transposon junctions show that population diversity is significantly reduced after selection due to preferential expansion of clones with multiple integration events. This non-viral, positive selection approach is an attractive alternative for producing engineered cells.

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The SB transposon system combined with chemical-induced dimerization (CID). (A) Constructs used in this study. pT3/eGIF plasmid carries the SB transposon containing of the EF1α promoter, eGFP gene, Internal Ribosome Entry Site (IRES) and a fusion gene of synthetic dimerization domain F36V and a Fibroblast Growth Factor Receptor 1 (FGFR1), positioned between Inverted Terminal Repeats (ITRs, black arrows); mcT3/eGIF is the minicircle derived from pT3/eGIF and SB100X plasmid contains the hyperactive SB transposase SB100X under the CMV promoter. (B) CID (AP20187) selection for cells with integrated transposon. Transfected cells expressing the F36VFGFR1 protein undergo selective proliferation through CID-induced cell signaling. After selection, a nearly homogenous population of stably transduced cells is obtained.
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gks213-F1: The SB transposon system combined with chemical-induced dimerization (CID). (A) Constructs used in this study. pT3/eGIF plasmid carries the SB transposon containing of the EF1α promoter, eGFP gene, Internal Ribosome Entry Site (IRES) and a fusion gene of synthetic dimerization domain F36V and a Fibroblast Growth Factor Receptor 1 (FGFR1), positioned between Inverted Terminal Repeats (ITRs, black arrows); mcT3/eGIF is the minicircle derived from pT3/eGIF and SB100X plasmid contains the hyperactive SB transposase SB100X under the CMV promoter. (B) CID (AP20187) selection for cells with integrated transposon. Transfected cells expressing the F36VFGFR1 protein undergo selective proliferation through CID-induced cell signaling. After selection, a nearly homogenous population of stably transduced cells is obtained.

Mentions: The pT3/eGIF plasmid that carries the hyperactive transposon (T3) cassette containing both the EGFP reporter gene and the F36VFGFR-1 gene expressed under the EF1α (elongation factor-1α) promoter through an internal ribosome entry sequence (IRES) was constructed (Figure 1). The hyperactive transposon, developed by Yant et al., contains a duplication of the left IR/DR structure in the transposon along with additional flanking TA dinucleotides and has been shown to be three times more active than the original transposon sequences (25). The analogous plasmid (pT3/mGIF, 8.3 kb) with the murine stem cell virus (MSCV) promoter was also constructed. These two promoter sequences were selected because they have both been shown to drive strong expression across different types of cells, including stem cells (26,27) The EF1α promoter drove more robust transgene expression (4–5 times) than the MSCV promoter in Ba/F3 cells (data not shown) so the EF1α promoter construct was used for all remaining studies.Figure 1.


Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells.

Kacherovsky N, Harkey MA, Blau CA, Giachelli CM, Pun SH - Nucleic Acids Res. (2012)

The SB transposon system combined with chemical-induced dimerization (CID). (A) Constructs used in this study. pT3/eGIF plasmid carries the SB transposon containing of the EF1α promoter, eGFP gene, Internal Ribosome Entry Site (IRES) and a fusion gene of synthetic dimerization domain F36V and a Fibroblast Growth Factor Receptor 1 (FGFR1), positioned between Inverted Terminal Repeats (ITRs, black arrows); mcT3/eGIF is the minicircle derived from pT3/eGIF and SB100X plasmid contains the hyperactive SB transposase SB100X under the CMV promoter. (B) CID (AP20187) selection for cells with integrated transposon. Transfected cells expressing the F36VFGFR1 protein undergo selective proliferation through CID-induced cell signaling. After selection, a nearly homogenous population of stably transduced cells is obtained.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367214&req=5

gks213-F1: The SB transposon system combined with chemical-induced dimerization (CID). (A) Constructs used in this study. pT3/eGIF plasmid carries the SB transposon containing of the EF1α promoter, eGFP gene, Internal Ribosome Entry Site (IRES) and a fusion gene of synthetic dimerization domain F36V and a Fibroblast Growth Factor Receptor 1 (FGFR1), positioned between Inverted Terminal Repeats (ITRs, black arrows); mcT3/eGIF is the minicircle derived from pT3/eGIF and SB100X plasmid contains the hyperactive SB transposase SB100X under the CMV promoter. (B) CID (AP20187) selection for cells with integrated transposon. Transfected cells expressing the F36VFGFR1 protein undergo selective proliferation through CID-induced cell signaling. After selection, a nearly homogenous population of stably transduced cells is obtained.
Mentions: The pT3/eGIF plasmid that carries the hyperactive transposon (T3) cassette containing both the EGFP reporter gene and the F36VFGFR-1 gene expressed under the EF1α (elongation factor-1α) promoter through an internal ribosome entry sequence (IRES) was constructed (Figure 1). The hyperactive transposon, developed by Yant et al., contains a duplication of the left IR/DR structure in the transposon along with additional flanking TA dinucleotides and has been shown to be three times more active than the original transposon sequences (25). The analogous plasmid (pT3/mGIF, 8.3 kb) with the murine stem cell virus (MSCV) promoter was also constructed. These two promoter sequences were selected because they have both been shown to drive strong expression across different types of cells, including stem cells (26,27) The EF1α promoter drove more robust transgene expression (4–5 times) than the MSCV promoter in Ba/F3 cells (data not shown) so the EF1α promoter construct was used for all remaining studies.Figure 1.

Bottom Line: The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern.Copy number analysis by quantitative PCR (qPCR) revealed that CID-selected cells contain on average higher copy numbers of transgenes than flow cytometry-selected cells, demonstrating selective advantage for cells with multiple transposon insertions.This non-viral, positive selection approach is an attractive alternative for producing engineered cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture times. Here, we introduce an efficient non-viral approach combining the Sleeping Beauty (SB) Transposon System with selective proliferation of engineered cells by chemically induced dimerization (CID) of growth factor receptors. Minicircles carrying a SB transposon cassette containing a reporter transgene and a gene for the F36VFGFR1 fusion protein were delivered to the hematopoietic cell line Ba/F3. Stably-transduced Ba/F3 cell populations with >98% purity were obtained within 1 week using this positive selection strategy. Copy number analysis by quantitative PCR (qPCR) revealed that CID-selected cells contain on average higher copy numbers of transgenes than flow cytometry-selected cells, demonstrating selective advantage for cells with multiple transposon insertions. A diverse population of cells is present both before and after culture in CID media, although site-specific qPCR of transposon junctions show that population diversity is significantly reduced after selection due to preferential expansion of clones with multiple integration events. This non-viral, positive selection approach is an attractive alternative for producing engineered cells.

Show MeSH
Related in: MedlinePlus