For tet-on DNMT1 systems, we synthesized the coding sequence of pDNMT1 gene from GENEWIZ by chemical method. The amplified sequence pDNMT1 was then cloned into a pFU-tetO lentivirus backbone (19778, Addgene) linearizing with EcoR1 restriction enzyme. The FUdeltaGW-rtTA (19780) and third-generation lentiviral helper plasmid (12253, 12252, 12251) were purchased from Addgene. pFU-tetO-pDNMT1 and FUdeltaGW-rtTA were co-transfected into MSCs. Plasmids with GFP genes were used as control. Because there was almost no significant differences between nsPEFs with the two set parameters (10 ns at 20 kV/cm, and 100 ns at 10 kV/cm), nsPEFs of 100 ns at 10 kV/cm was used for studying the effects of downregulation of DNMT1. After stimulation by nsPEFs, doxycycline (Dox) was added to MSCs at 1 μg/ml for 2 h. The expression levels of GFP and DNMT1 were evaluated by western blotting. The primers and annealing temperatures used for PCR of GFP and DNMT1 are listed in Supplementary Table 3 . The experiment was repeated three times, with five technological repeats for each assay.
Pfu teto lentivirus backbone
Manufactured by Addgene
The PFU-tetO lentivirus backbone is a genetic construct designed for the expression of genes under the control of the tetracycline-responsive promoter (tetO). This backbone can be used to create lentiviral particles for the delivery and regulation of gene expression in target cells.
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Lab products found in correlation
3 protocols using pfu teto lentivirus backbone
1
Inducible DNMT1 Expression in MSCs
2
Inducible DNMT1 Modulation in MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For tet-on DNMT1 systems, we synthesized the coding sequence of pDNMT1 gene from GENEWIZ by chemical method. The ampli ed sequence pDNMT1 was then cloned into a pFU-tetO lentivirus backbone (19778, Addgene) linearizing with EcoR1 restriction enzyme. The FUdeltaGW-rtTA (19780) and third generation lentiviral helper plasmid (12253, 12252, 12251) were purchased from Addgene. pFU-tetO-pDNMT1 and FUdeltaGW-rtTA were co-transfected into MSCs. Plasmids with GFP genes were used as control. Because there was almost no signi cantly differences between nsPEFs with the two set parameters (10 ns at 20 kV/cm, and 100 ns at 10 kV/cm), nsPEFs of 100 ns at 10 kV/cm was used for studying the effects of downregulation of DNMT1. After stimulation by nsPEFs, doxycycline (Dox) were added to MSCs at 1 μg/ml for 2 hours. The expression levels of GFP and DNMT1 were evaluated by western blotting. The primers and annealing temperatures used for PCR of GFP and DNMT1 are listed in Supplementary Table 3. The experiment was repeated for three times, with ve technological repeats for each assay.
3
Inducible DNMT1 Expression in MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For tet-on DNMT1 systems, we synthesized the coding sequence of pDNMT1 gene from GENEWIZ by chemical method. The ampli ed sequence pDNMT1 was then cloned into a pFU-tetO lentivirus backbone (19778, Addgene) linearizing with EcoR1 restriction enzyme. The FUdeltaGW-rtTA (19780) and third generation lentiviral helper plasmid (12253, 12252, 12251) were purchased from Addgene. pFU-tetO-pDNMT1 and FUdeltaGW-rtTA were co-transfected into MSCs. Plasmids with GFP genes were used as control. Because there was almost no signi cantly differences between nsPEFs with the two set parameters (10 ns at 20 kV/cm, and 100 ns at 10 kV/cm), nsPEFs of 100 ns at 10 kV/cm was used for studying the effects of downregulation of DNMT1. After stimulation by nsPEFs, doxycycline (Dox) were added to MSCs at 1 µg/ml for 2 hours. The expression levels of GFP and DNMT1 were evaluated by western blotting. The primers and annealing temperatures used for PCR of GFP and DNMT1 are listed in Supplementary Table 3. The experiment was repeated for three times, with ve technological repeats for each assay.
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