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Addgene inc plasmid xlox tert pgk icasp9 ires gfp

Retroviral transduction of NK cells with hTERT or/and <t>iCasp9</t> genes. ( A ) The ACTB normalized expression levels of hTERT transgene evaluated by qPCR analysis ( n = 7). * p < 0.05. ( B ) The ACTB normalized expression levels of iCasp9 transgene evaluated by qPCR analysis ( n = 7). Friedman test with Dunn’s multiple comparison test, median, p -value: * p < 0.05; ** p < 0.01. ( C ) Telomerase activity in modified NK cells determined by TRAP assay for qPCR method ( n = 4). The K562 cell line was used as positive control. For comparison, data were divided into 2 groups (1st with hTERT transgenes: hTERT + with hTERT + iCasp9 + ; 2nd without hTERT transgenes: untransduced with iCasp9 + ). Mann–Whitney test, median, p -value: * p < 0.05. ( D ) Proportion of dead NK cells treated for 24 h with 100 nM chemical inductor of dimerization (CID) ( n = 6). NK cells cultured without CID were used as a control. Dead cells were determined as positive for AnnexinV and/or SYTOX staining. The increase in percentage of dead NK cells was calculated by the following formula: Δ% = 100 nM CID-treated dead NK cells %—dead NK cells in control %. Ordinary one-way ANOVA with Tukey’s multiple comparison test, mean, p -value: * p < 0.05; ** p < 0.01.

Plasmid Xlox Tert Pgk Icasp9 Ires Gfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "The hTERT and iCasp9 Transgenes Affect EOMES and T-BET Levels in NK Cells and the Introduction of Both Genes Improves NK Cell Proliferation in Response to IL2 and IL15 Stimulation"

Article Title: The hTERT and iCasp9 Transgenes Affect EOMES and T-BET Levels in NK Cells and the Introduction of Both Genes Improves NK Cell Proliferation in Response to IL2 and IL15 Stimulation

Journal: Biomedicines

doi: 10.3390/biomedicines12030650

Retroviral transduction of NK cells with hTERT or/and iCasp9 genes. ( A ) The ACTB normalized expression levels of hTERT transgene evaluated by qPCR analysis ( n = 7). * p < 0.05. ( B ) The ACTB normalized expression levels of iCasp9 transgene evaluated by qPCR analysis ( n = 7). Friedman test with Dunn’s multiple comparison test, median, p -value: * p < 0.05; ** p < 0.01. ( C ) Telomerase activity in modified NK cells determined by TRAP assay for qPCR method ( n = 4). The K562 cell line was used as positive control. For comparison, data were divided into 2 groups (1st with hTERT transgenes: hTERT + with hTERT + iCasp9 + ; 2nd without hTERT transgenes: untransduced with iCasp9 + ). Mann–Whitney test, median, p -value: * p < 0.05. ( D ) Proportion of dead NK cells treated for 24 h with 100 nM chemical inductor of dimerization (CID) ( n = 6). NK cells cultured without CID were used as a control. Dead cells were determined as positive for AnnexinV and/or SYTOX staining. The increase in percentage of dead NK cells was calculated by the following formula: Δ% = 100 nM CID-treated dead NK cells %—dead NK cells in control %. Ordinary one-way ANOVA with Tukey’s multiple comparison test, mean, p -value: * p < 0.05; ** p < 0.01.

Figure Legend Snippet: Retroviral transduction of NK cells with hTERT or/and iCasp9 genes. ( A ) The ACTB normalized expression levels of hTERT transgene evaluated by qPCR analysis ( n = 7). * p < 0.05. ( B ) The ACTB normalized expression levels of iCasp9 transgene evaluated by qPCR analysis ( n = 7). Friedman test with Dunn’s multiple comparison test, median, p -value: * p < 0.05; ** p < 0.01. ( C ) Telomerase activity in modified NK cells determined by TRAP assay for qPCR method ( n = 4). The K562 cell line was used as positive control. For comparison, data were divided into 2 groups (1st with hTERT transgenes: hTERT + with hTERT + iCasp9 + ; 2nd without hTERT transgenes: untransduced with iCasp9 + ). Mann–Whitney test, median, p -value: * p < 0.05. ( D ) Proportion of dead NK cells treated for 24 h with 100 nM chemical inductor of dimerization (CID) ( n = 6). NK cells cultured without CID were used as a control. Dead cells were determined as positive for AnnexinV and/or SYTOX staining. The increase in percentage of dead NK cells was calculated by the following formula: Δ% = 100 nM CID-treated dead NK cells %—dead NK cells in control %. Ordinary one-way ANOVA with Tukey’s multiple comparison test, mean, p -value: * p < 0.05; ** p < 0.01.

Techniques Used: Retroviral, Transduction, Expressing, Comparison, Activity Assay, Modification, TRAP Assay, Positive Control, MANN-WHITNEY, Cell Culture, Control, Staining

Proliferation assay started 1 month after ex vivo isolation of NK cells modified with hTERT and/or iCasp9 transgenes. Stimulation with IL2, IL2+K562-mbIL21 and IL2+IL15 was performed. ( A ) Numbers of NK cells cultured for one month with IL2+IL15. Mixed effects analysis, Sidak’s multiple comparisons test, n = 5, median, p -value: * p < 0.05. For IL2, IL2+K562-mbIL21 and IL2+IL15 types of stimulations: ( B ) the proportion of live NK cells and ( C ) the weekly increase n (week+1) /N week are presented. Vertical dashed line stands for a time point of 2 months after isolation. Friedman test with Dunn’s multiple comparisons, n = 7, median, p -value: * p < 0.05; ** p < 0.01; *** p < 0.001. ( D ) The proportions of live NK cells for each type of stimulation in 2 months of ex vivo isolation. ( E ) The comparison of expansion coefficient between IL2, IL2+K562-mbIL21 and IL2+IL15 stimulations in 2 months of ex vivo isolation. Friedman test with Dunn’s multiple comparisons, median, n = 5, p -value: * p < 0.05; ** p < 0.01.

Figure Legend Snippet: Proliferation assay started 1 month after ex vivo isolation of NK cells modified with hTERT and/or iCasp9 transgenes. Stimulation with IL2, IL2+K562-mbIL21 and IL2+IL15 was performed. ( A ) Numbers of NK cells cultured for one month with IL2+IL15. Mixed effects analysis, Sidak’s multiple comparisons test, n = 5, median, p -value: * p < 0.05. For IL2, IL2+K562-mbIL21 and IL2+IL15 types of stimulations: ( B ) the proportion of live NK cells and ( C ) the weekly increase n (week+1) /N week are presented. Vertical dashed line stands for a time point of 2 months after isolation. Friedman test with Dunn’s multiple comparisons, n = 7, median, p -value: * p < 0.05; ** p < 0.01; *** p < 0.001. ( D ) The proportions of live NK cells for each type of stimulation in 2 months of ex vivo isolation. ( E ) The comparison of expansion coefficient between IL2, IL2+K562-mbIL21 and IL2+IL15 stimulations in 2 months of ex vivo isolation. Friedman test with Dunn’s multiple comparisons, median, n = 5, p -value: * p < 0.05; ** p < 0.01.

Techniques Used: Proliferation Assay, Ex Vivo, Isolation, Modification, Cell Culture, Comparison

The expression of EOMES and T-BET transcription factors in NK cells modified with hTERT and/or iCasp9 genes. ( A ) NK cells cultivated with IL2+K562-mbIL21 are represented. A change in the proportion of EOMES- and T-BET-expressing cells in 1st and to 2nd month after ex vivo isolation (N ex vivo = 8, N 1 month = 5, N 2 months = 2, mean). ( B ) The distribution of EOMES +/− T-BET +/− subsets of NK cells cultured for 1 month. EOMES − T-BET − , EOMES + T-BET + (for comparison, data were divided into 2 groups (1st: hTERT + with hTERT + iCasp9 + ; 2nd: untransduced with iCasp9 + )), EOMES + T-BET − (for comparison, data were divided into 2 groups (1st: untransduced with hTERT + ; 2nd: hTERT + iCasp9 + with iCasp9 + )), EOMES − T-BET + hTERT and/or iCasp9 modified NK cells a month after ex vivo isolation ( n = 5). Unpaired t -test, mean, p -value: * p < 0.05. ( C ) Representative density plots for EOMES and T-BET distributions in NK cells ex vivo, 1 month and 2 months after isolation.

Figure Legend Snippet: The expression of EOMES and T-BET transcription factors in NK cells modified with hTERT and/or iCasp9 genes. ( A ) NK cells cultivated with IL2+K562-mbIL21 are represented. A change in the proportion of EOMES- and T-BET-expressing cells in 1st and to 2nd month after ex vivo isolation (N ex vivo = 8, N 1 month = 5, N 2 months = 2, mean). ( B ) The distribution of EOMES +/− T-BET +/− subsets of NK cells cultured for 1 month. EOMES − T-BET − , EOMES + T-BET + (for comparison, data were divided into 2 groups (1st: hTERT + with hTERT + iCasp9 + ; 2nd: untransduced with iCasp9 + )), EOMES + T-BET − (for comparison, data were divided into 2 groups (1st: untransduced with hTERT + ; 2nd: hTERT + iCasp9 + with iCasp9 + )), EOMES − T-BET + hTERT and/or iCasp9 modified NK cells a month after ex vivo isolation ( n = 5). Unpaired t -test, mean, p -value: * p < 0.05. ( C ) Representative density plots for EOMES and T-BET distributions in NK cells ex vivo, 1 month and 2 months after isolation.

Techniques Used: Expressing, Modification, Ex Vivo, Isolation, Cell Culture, Comparison

The proportions of NK cells modified with hTERT and/or iCasp9 genes 2 months after ex vivo isolation. Data for IL2, IL2+K562-mbIL21 and IL2+IL15 stimulated NK cells are represented. ( A ) The expression levels of immune checkpoints TIM-3, TIGIT and KLGR-1. Dotted boxes stand to determine the groups compared. Two-way ANOVA with Tukey’s multiple comparisons test, n = 3, mean, p -value: * p < 0.05; ** p < 0.01; *** p < 0.001. ( B ) Surface expression (means) of CD16, KIR (KIR2DL2/DL3), PD-1, NKG2C, CD57, CD56, NKG2A, HLA-DR, NKp30, NKp44 and NKp46 for NK cells stimulated by IL2+K562-mbIL21.

Figure Legend Snippet: The proportions of NK cells modified with hTERT and/or iCasp9 genes 2 months after ex vivo isolation. Data for IL2, IL2+K562-mbIL21 and IL2+IL15 stimulated NK cells are represented. ( A ) The expression levels of immune checkpoints TIM-3, TIGIT and KLGR-1. Dotted boxes stand to determine the groups compared. Two-way ANOVA with Tukey’s multiple comparisons test, n = 3, mean, p -value: * p < 0.05; ** p < 0.01; *** p < 0.001. ( B ) Surface expression (means) of CD16, KIR (KIR2DL2/DL3), PD-1, NKG2C, CD57, CD56, NKG2A, HLA-DR, NKp30, NKp44 and NKp46 for NK cells stimulated by IL2+K562-mbIL21.

Techniques Used: Modification, Ex Vivo, Isolation, Expressing

The dynamics of normalized mRNA expression levels of the EOMES and TBX21 genes encoding transcription factors EOMES and T-BET along with expression levels of pro-survival BCL2 , MCL1 , BCL2L1 (BCL-X L ) and BIRC5 , pro-apoptotic BAX , BAD , BAK , DIABLO and BBC3 (PUMA), immune-checkpoint-encoding genes TIM3 , TIGIT and LAG3 and exhaustion-associated SOCS1-3 and CISH . Data obtained at time points of 1 month (1 m) and 2 months (2 m) after ex vivo isolation of hTERT and/or iCasp9 modified NK cells cultured with IL2+K562-mbIL21. Dotted boxes stand to highlight groups compared. Mann–Whitney, N1m = 10, N2m = 3, median, p -value: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure Legend Snippet: The dynamics of normalized mRNA expression levels of the EOMES and TBX21 genes encoding transcription factors EOMES and T-BET along with expression levels of pro-survival BCL2 , MCL1 , BCL2L1 (BCL-X L ) and BIRC5 , pro-apoptotic BAX , BAD , BAK , DIABLO and BBC3 (PUMA), immune-checkpoint-encoding genes TIM3 , TIGIT and LAG3 and exhaustion-associated SOCS1-3 and CISH . Data obtained at time points of 1 month (1 m) and 2 months (2 m) after ex vivo isolation of hTERT and/or iCasp9 modified NK cells cultured with IL2+K562-mbIL21. Dotted boxes stand to highlight groups compared. Mann–Whitney, N1m = 10, N2m = 3, median, p -value: * p < 0.05; ** p < 0.01; *** p < 0.001.

Techniques Used: Expressing, Ex Vivo, Isolation, Modification, Cell Culture, MANN-WHITNEY

$The functional activity of NK cells modified with hTERT and/or iCasp9 genes a month after their ex vivo isolation. ( A ) The proportion of NK cells accumulating IFNγ in response to IL2, IL12 and IL18 cytokines (Top). The intensity of cytokine-dependent IFNγ production per cell measured by mean fluorescence intensity normalized to FMO control (bottom). ( B ) The fraction of NK cells capable of degranulation measured by LAMP-1 surface exposure in response to the recognition of K562 target cells (Top). The intensity of degranulation in response to K562 cells production per cell is measured by mean fluorescence intensity normalized to basal degranulation (bottom). The cells were also divided into groups I (IFNγ > 50% and MFI LAMP-1 < 5) and II (IFNγ < 50% and MFI LAMP-1 > 5). ( C ) The donor dependency of IFNγ production. Ordinary one-way ANOVA, n = 7 donors.$
Figure Legend Snippet: The functional activity of NK cells modified with hTERT and/or iCasp9 genes a month after their ex vivo isolation. ( A ) The proportion of NK cells accumulating IFNγ in response to IL2, IL12 and IL18 cytokines (Top). The intensity of cytokine-dependent IFNγ production per cell measured by mean fluorescence intensity normalized to FMO control (bottom). ( B ) The fraction of NK cells capable of degranulation measured by LAMP-1 surface exposure in response to the recognition of K562 target cells (Top). The intensity of degranulation in response to K562 cells production per cell is measured by mean fluorescence intensity normalized to basal degranulation (bottom). The cells were also divided into groups I (IFNγ > 50% and MFI LAMP-1 < 5) and II (IFNγ < 50% and MFI LAMP-1 > 5). ( C ) The donor dependency of IFNγ production. Ordinary one-way ANOVA, n = 7 donors.

Techniques Used: Functional Assay, Activity Assay, Modification, Ex Vivo, Isolation, Fluorescence, Control

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