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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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Transient and stable transfection of Ba/F3 cells using SB transposon system. (A) Comparison of transient transfection efficiency by nucleofection delivery of plasmid and minicircle bearing T3/eGIF transposon. Ba/F3 cells were nucleofected with 5 µg of either plasmid pT3/eGIF or minicircle mcT3/eGIF. Flow cytometry was performed 24 h after nucleofection. (B) Integration of T3/eGIF transposon. Ba/F3 cells were nucleofected with varying DNA amounts of transposon minicircle mcT3/eGIF and transposase plasmid pSB100x. Flow cytometry was performed 24 h (gray bars) and 7 days (striped bars) after nucleofection to assess transient and stable transfection efficiency, respectively. Bars represent SD of three independent transfections. Averages of mean fluorescence intensity of GFP+/PI− populations are indicated above the graphs in RFU.
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gks213-F2: Transient and stable transfection of Ba/F3 cells using SB transposon system. (A) Comparison of transient transfection efficiency by nucleofection delivery of plasmid and minicircle bearing T3/eGIF transposon. Ba/F3 cells were nucleofected with 5 µg of either plasmid pT3/eGIF or minicircle mcT3/eGIF. Flow cytometry was performed 24 h after nucleofection. (B) Integration of T3/eGIF transposon. Ba/F3 cells were nucleofected with varying DNA amounts of transposon minicircle mcT3/eGIF and transposase plasmid pSB100x. Flow cytometry was performed 24 h (gray bars) and 7 days (striped bars) after nucleofection to assess transient and stable transfection efficiency, respectively. Bars represent SD of three independent transfections. Averages of mean fluorescence intensity of GFP+/PI− populations are indicated above the graphs in RFU.

Mentions: Transfection studies were performed in the IL-3 dependent, FGFR-1 responsive pro-B cell line Ba/F3 using nucleofection for gene delivery. The efficiency of nucleofection was 4 times higher for minicircles carrying transposon, than the pT3/eGIF plasmid, when equal amount of the DNA was used (Figure 2A). The mean GFP fluorescence of the transfected population is similar for both minicircle- and plasmid-transfected cells, indicating that expression efficiency is similar between the two constructs. No difference in cell viability after nucleofection was observed as well (data not shown). Next, the transposon integration efficiency was tested by codelivery of mcT3/eGIF with the SB100X transposase plasmid by flow cytometry analysis for EGFP-positive cells 24 h and 7 days post-nucleofection to assess for efficiency of transient and stable expression, respectively. By Day 7 post-nucleofection, delivered transposon in the absence of transposase was eliminated, resulting in 0.1% EGFP+ cells. In contrast, ∼5% of live cells (or 25% of transiently transfected population) carried integrated transposons due to codelivery of transposase (Figure 2B). Increasing the transposon to transposase plasmids ratio from 1:2 to 1:1 (by mass) did not significantly affect transposition efficiency.Figure 2.


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)

Transient and stable transfection of Ba/F3 cells using SB transposon system. (A) Comparison of transient transfection efficiency by nucleofection delivery of plasmid and minicircle bearing T3/eGIF transposon. Ba/F3 cells were nucleofected with 5 µg of either plasmid pT3/eGIF or minicircle mcT3/eGIF. Flow cytometry was performed 24 h after nucleofection. (B) Integration of T3/eGIF transposon. Ba/F3 cells were nucleofected with varying DNA amounts of transposon minicircle mcT3/eGIF and transposase plasmid pSB100x. Flow cytometry was performed 24 h (gray bars) and 7 days (striped bars) after nucleofection to assess transient and stable transfection efficiency, respectively. Bars represent SD of three independent transfections. Averages of mean fluorescence intensity of GFP+/PI− populations are indicated above the graphs in RFU.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks213-F2: Transient and stable transfection of Ba/F3 cells using SB transposon system. (A) Comparison of transient transfection efficiency by nucleofection delivery of plasmid and minicircle bearing T3/eGIF transposon. Ba/F3 cells were nucleofected with 5 µg of either plasmid pT3/eGIF or minicircle mcT3/eGIF. Flow cytometry was performed 24 h after nucleofection. (B) Integration of T3/eGIF transposon. Ba/F3 cells were nucleofected with varying DNA amounts of transposon minicircle mcT3/eGIF and transposase plasmid pSB100x. Flow cytometry was performed 24 h (gray bars) and 7 days (striped bars) after nucleofection to assess transient and stable transfection efficiency, respectively. Bars represent SD of three independent transfections. Averages of mean fluorescence intensity of GFP+/PI− populations are indicated above the graphs in RFU.
Mentions: Transfection studies were performed in the IL-3 dependent, FGFR-1 responsive pro-B cell line Ba/F3 using nucleofection for gene delivery. The efficiency of nucleofection was 4 times higher for minicircles carrying transposon, than the pT3/eGIF plasmid, when equal amount of the DNA was used (Figure 2A). The mean GFP fluorescence of the transfected population is similar for both minicircle- and plasmid-transfected cells, indicating that expression efficiency is similar between the two constructs. No difference in cell viability after nucleofection was observed as well (data not shown). Next, the transposon integration efficiency was tested by codelivery of mcT3/eGIF with the SB100X transposase plasmid by flow cytometry analysis for EGFP-positive cells 24 h and 7 days post-nucleofection to assess for efficiency of transient and stable expression, respectively. By Day 7 post-nucleofection, delivered transposon in the absence of transposase was eliminated, resulting in 0.1% EGFP+ cells. In contrast, ∼5% of live cells (or 25% of transiently transfected population) carried integrated transposons due to codelivery of transposase (Figure 2B). Increasing the transposon to transposase plasmids ratio from 1:2 to 1:1 (by mass) did not significantly affect transposition efficiency.Figure 2.

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