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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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Proliferation of transfected Ba/F3 cells cultured in IL-3 or CID-containing media. (A) Flow cytometry analysis of stably transfected populations of BaF3 cells with T3/eGIF transposon, grown in the presence of CID (AP20187) or IL-3 at different time points. (B) Growth curve of untransfected (WT) or SD transfected (T3/eGIF) Ba/F3 cells cultured in either IL-3 or CID (AP20187) containing media. An arrow indicates time (Day 0) when CID selection commenced. Bars represent SDs of three independent cultures.
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gks213-F3: Proliferation of transfected Ba/F3 cells cultured in IL-3 or CID-containing media. (A) Flow cytometry analysis of stably transfected populations of BaF3 cells with T3/eGIF transposon, grown in the presence of CID (AP20187) or IL-3 at different time points. (B) Growth curve of untransfected (WT) or SD transfected (T3/eGIF) Ba/F3 cells cultured in either IL-3 or CID (AP20187) containing media. An arrow indicates time (Day 0) when CID selection commenced. Bars represent SDs of three independent cultures.

Mentions: Pools of Ba/F3 cells with a starting integrated transposon frequency of ∼5% were cultured in the presence of 100 nM AP20187 in IL3-free conditions to allow for CID selection. Ba/F3 cells are normally IL-3 dependent. The population of transformed cells was monitored by flow cytometry at 2, 4 and 7 days post-selection. The percentage of EGFP+ cells in the live cell population (negative for propidium iodide staining) increased steadily, reaching >98% with 7 days of CID selection (Figure 3A). Transformed cells maintained in IL-3 kept a stable population of 5.6 ± 1.8% of EGFP+ cells (Figure 3A). The growth kinetics of transformed Ba/F3 cells grown in IL3 and CID was also determined (Figure 3B). Control (untransfected) Ba/F3 and transfected Ba/F3 cells showed similar growth curves when cultured in IL-3 containing media. Transfected Ba/F3 cells decreased in population 2 days after transitioning to CID-containing, IL-3-deficient media due to death of untransformed cells. However, cell numbers began to increase at 4 days post-selection and continued to expand with CID-induced proliferation. The doubling time of transformed Ba/F3 cells under CID expansion was similar as control cells growing in IL-3 media (doubling time of 19–23 h, data not shown). As expected, control Ba/F3 cells were unable to survive in the absence of IL-3.Figure 3.


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)

Proliferation of transfected Ba/F3 cells cultured in IL-3 or CID-containing media. (A) Flow cytometry analysis of stably transfected populations of BaF3 cells with T3/eGIF transposon, grown in the presence of CID (AP20187) or IL-3 at different time points. (B) Growth curve of untransfected (WT) or SD transfected (T3/eGIF) Ba/F3 cells cultured in either IL-3 or CID (AP20187) containing media. An arrow indicates time (Day 0) when CID selection commenced. Bars represent SDs of three independent cultures.
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gks213-F3: Proliferation of transfected Ba/F3 cells cultured in IL-3 or CID-containing media. (A) Flow cytometry analysis of stably transfected populations of BaF3 cells with T3/eGIF transposon, grown in the presence of CID (AP20187) or IL-3 at different time points. (B) Growth curve of untransfected (WT) or SD transfected (T3/eGIF) Ba/F3 cells cultured in either IL-3 or CID (AP20187) containing media. An arrow indicates time (Day 0) when CID selection commenced. Bars represent SDs of three independent cultures.
Mentions: Pools of Ba/F3 cells with a starting integrated transposon frequency of ∼5% were cultured in the presence of 100 nM AP20187 in IL3-free conditions to allow for CID selection. Ba/F3 cells are normally IL-3 dependent. The population of transformed cells was monitored by flow cytometry at 2, 4 and 7 days post-selection. The percentage of EGFP+ cells in the live cell population (negative for propidium iodide staining) increased steadily, reaching >98% with 7 days of CID selection (Figure 3A). Transformed cells maintained in IL-3 kept a stable population of 5.6 ± 1.8% of EGFP+ cells (Figure 3A). The growth kinetics of transformed Ba/F3 cells grown in IL3 and CID was also determined (Figure 3B). Control (untransfected) Ba/F3 and transfected Ba/F3 cells showed similar growth curves when cultured in IL-3 containing media. Transfected Ba/F3 cells decreased in population 2 days after transitioning to CID-containing, IL-3-deficient media due to death of untransformed cells. However, cell numbers began to increase at 4 days post-selection and continued to expand with CID-induced proliferation. The doubling time of transformed Ba/F3 cells under CID expansion was similar as control cells growing in IL-3 media (doubling time of 19–23 h, data not shown). As expected, control Ba/F3 cells were unable to survive in the absence of IL-3.Figure 3.

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