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A diphtheria toxin resistance marker for in vitro and in vivo selection of stably transduced human cells.

Picco G, Petti C, Trusolino L, Bertotti A, Medico E - Sci Rep (2015)

Bottom Line: DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth.DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers.This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.

View Article: PubMed Central - PubMed

Affiliation: Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Torino, Italy.

ABSTRACT
We developed a selectable marker rendering human cells resistant to Diphtheria Toxin (DT). The marker (DT(R)) consists of a primary microRNA sequence engineered to downregulate the ubiquitous DPH2 gene, a key enzyme for the biosynthesis of the DT target diphthamide. DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth. DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers. As mice are insensitive to DT, DT(R)-based selection can be also applied in vivo. Direct injection of a GFP-DT(R) lentiviral vector into human cancer cell-line xenografts and patient-derived tumorgrafts implanted in mice, followed by systemic DT administration, yielded tumors entirely composed of permanently transduced cells and detectable by imaging systems. This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.

No MeSH data available.


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DPH2 silencing renders human cell lines resistant to DT.(a) Crystal violet staining of HCT116 cells transduced with Scramble or shRNAmirs targeting DPH2 (sh1-4), DPH5 (sh5-9), DPH6 (sh10-13) and DPH7 (sh14-18), grown in the absence or presence of DT (10 ng/ml) for one week. The only construct conferring resistance to DT was sh4, hence renamed DTR. (b) Crystal violet staining of human normal (HME1) and neoplastic cell lines (HCT116, colon; A549, lung; OVCAR4, ovary; SNB19, glioblastoma) transduced with Scramble or DTR vector and grown in the absence or presence of DT (1 ng/ml or 10 ng/ml) for one week.
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f1: DPH2 silencing renders human cell lines resistant to DT.(a) Crystal violet staining of HCT116 cells transduced with Scramble or shRNAmirs targeting DPH2 (sh1-4), DPH5 (sh5-9), DPH6 (sh10-13) and DPH7 (sh14-18), grown in the absence or presence of DT (10 ng/ml) for one week. The only construct conferring resistance to DT was sh4, hence renamed DTR. (b) Crystal violet staining of human normal (HME1) and neoplastic cell lines (HCT116, colon; A549, lung; OVCAR4, ovary; SNB19, glioblastoma) transduced with Scramble or DTR vector and grown in the absence or presence of DT (1 ng/ml or 10 ng/ml) for one week.

Mentions: To validate DT as a selective agent, we verified its activity on a panel of cell lines of different tissue origin, expressing variable levels of HBEGF23. DT activity was indeed always high and independent of HBEGF expression levels (Supplementary Fig. 2). To assess if silencing of the genes involved in diphthamide biosynthesis confers resistance to DT, HCT116 colorectal cancer (CRC) cells, expressing intermediate levels of HBEGF, were chosen as a model and transduced with 18 different shRNAmir constructs, targeting the DPH2, DPH5, DPH6 and DPH7 transcripts (Supplementary Table 1). Transduced cells were then tested for their response to DT (Fig. 1a) and for downregulation of the target transcript (Supplementary Table 2). Among all the tested shRNAmirs, only the construct #4, targeting DPH2, was found to induce both robust downregulation of DPH2 mRNA levels and strong resistance to DT. This shRNAmir sequence is hereafter referred to as DTR (“diphtheria toxin resistance”). Efficient downregulation of DPH2 and induction of resistance to DT was confirmed in three additional human cancer cell lines derived from different tissues and in a non-transformed human breast epithelial cell line (Fig. 1b, Supplementary Table 2). Notably, cells expressing the DTR marker were not impaired in their growth rate (Supplementary Fig. 3). To assess if DTR can be efficiently employed as a selectable marker in vitro, we exploited the expression cassette for GFP and puromycin resistance included in the lentiviral DTR vector. HCT116 were transduced with DTR at low MOI to obtain around 2% of GFP-positive (GFP+) cells, and then selected in the presence of either DT (10 ng/ml) or puromycin (2 ng/ml) for two weeks. Both selections were found to strongly increase the GFP+ fraction (>95%; Supplementary Fig. 3). To verify the stability of the resistant phenotype, both selected populations were grown in the absence of DT or puromycin for one month, and found to retain a very high fraction of GFP+ cells (~80% and ~90% for puromycin and DT-selected cells, respectively; Supplementary Fig. 4). Persistence of the high GFP+ fraction was also observed after multiple freeze-thaw cycles (not shown).


A diphtheria toxin resistance marker for in vitro and in vivo selection of stably transduced human cells.

Picco G, Petti C, Trusolino L, Bertotti A, Medico E - Sci Rep (2015)

DPH2 silencing renders human cell lines resistant to DT.(a) Crystal violet staining of HCT116 cells transduced with Scramble or shRNAmirs targeting DPH2 (sh1-4), DPH5 (sh5-9), DPH6 (sh10-13) and DPH7 (sh14-18), grown in the absence or presence of DT (10 ng/ml) for one week. The only construct conferring resistance to DT was sh4, hence renamed DTR. (b) Crystal violet staining of human normal (HME1) and neoplastic cell lines (HCT116, colon; A549, lung; OVCAR4, ovary; SNB19, glioblastoma) transduced with Scramble or DTR vector and grown in the absence or presence of DT (1 ng/ml or 10 ng/ml) for one week.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588510&req=5

f1: DPH2 silencing renders human cell lines resistant to DT.(a) Crystal violet staining of HCT116 cells transduced with Scramble or shRNAmirs targeting DPH2 (sh1-4), DPH5 (sh5-9), DPH6 (sh10-13) and DPH7 (sh14-18), grown in the absence or presence of DT (10 ng/ml) for one week. The only construct conferring resistance to DT was sh4, hence renamed DTR. (b) Crystal violet staining of human normal (HME1) and neoplastic cell lines (HCT116, colon; A549, lung; OVCAR4, ovary; SNB19, glioblastoma) transduced with Scramble or DTR vector and grown in the absence or presence of DT (1 ng/ml or 10 ng/ml) for one week.
Mentions: To validate DT as a selective agent, we verified its activity on a panel of cell lines of different tissue origin, expressing variable levels of HBEGF23. DT activity was indeed always high and independent of HBEGF expression levels (Supplementary Fig. 2). To assess if silencing of the genes involved in diphthamide biosynthesis confers resistance to DT, HCT116 colorectal cancer (CRC) cells, expressing intermediate levels of HBEGF, were chosen as a model and transduced with 18 different shRNAmir constructs, targeting the DPH2, DPH5, DPH6 and DPH7 transcripts (Supplementary Table 1). Transduced cells were then tested for their response to DT (Fig. 1a) and for downregulation of the target transcript (Supplementary Table 2). Among all the tested shRNAmirs, only the construct #4, targeting DPH2, was found to induce both robust downregulation of DPH2 mRNA levels and strong resistance to DT. This shRNAmir sequence is hereafter referred to as DTR (“diphtheria toxin resistance”). Efficient downregulation of DPH2 and induction of resistance to DT was confirmed in three additional human cancer cell lines derived from different tissues and in a non-transformed human breast epithelial cell line (Fig. 1b, Supplementary Table 2). Notably, cells expressing the DTR marker were not impaired in their growth rate (Supplementary Fig. 3). To assess if DTR can be efficiently employed as a selectable marker in vitro, we exploited the expression cassette for GFP and puromycin resistance included in the lentiviral DTR vector. HCT116 were transduced with DTR at low MOI to obtain around 2% of GFP-positive (GFP+) cells, and then selected in the presence of either DT (10 ng/ml) or puromycin (2 ng/ml) for two weeks. Both selections were found to strongly increase the GFP+ fraction (>95%; Supplementary Fig. 3). To verify the stability of the resistant phenotype, both selected populations were grown in the absence of DT or puromycin for one month, and found to retain a very high fraction of GFP+ cells (~80% and ~90% for puromycin and DT-selected cells, respectively; Supplementary Fig. 4). Persistence of the high GFP+ fraction was also observed after multiple freeze-thaw cycles (not shown).

Bottom Line: DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth.DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers.This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.

View Article: PubMed Central - PubMed

Affiliation: Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Torino, Italy.

ABSTRACT
We developed a selectable marker rendering human cells resistant to Diphtheria Toxin (DT). The marker (DT(R)) consists of a primary microRNA sequence engineered to downregulate the ubiquitous DPH2 gene, a key enzyme for the biosynthesis of the DT target diphthamide. DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth. DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers. As mice are insensitive to DT, DT(R)-based selection can be also applied in vivo. Direct injection of a GFP-DT(R) lentiviral vector into human cancer cell-line xenografts and patient-derived tumorgrafts implanted in mice, followed by systemic DT administration, yielded tumors entirely composed of permanently transduced cells and detectable by imaging systems. This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.

No MeSH data available.


Related in: MedlinePlus