The human cDNA fragment encoding full-length ZEB1 and Ngn3 was prepared by PCR and cloned into pLV-EF1-MCS-IRES-Bsd (Biosettia, San Diego, USA). The lentiviral-based vector pLV-H1-EF1α-puro (Biosettia, San Diego, USA) was used to express shRNAs in breast cancer cells. The human Ngn3 full-length and truncated promoter sequences were obtained by PCR from human genomic DNA and cloned into the pGL3-basic vector (Promega, Wisconsin, USA). Primer sequences are listed in Supplementary Data.
Plv ef1 mcs ires bsd
Manufactured by Biosettia
Sourced in United States
The PLV-EF1-MCS-IRES-Bsd is a plasmid vector that contains the EF1 promoter, a multiple cloning site, an internal ribosome entry site, and a blasticidin resistance gene. This vector is commonly used for the expression of genes of interest in mammalian cell lines.
Automatically generated - may contain errors
6 protocols using plv ef1 mcs ires bsd
1
Cloning and Expression of ZEB1, Ngn3 in Breast Cancer
2
Cloning and Mutagenesis of ER-α Promoter
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The human complementary DNA fragment encoding full-length ZEB137 (link) was prepared by PCR and cloned into pLV-EF1-MCS-IRES-Bsd (Biosettia, San Diego, USA). The lentiviral-based vector pLV-H1-EF1α-puro (Biosettia, San Diego, USA) was used to express shRNAs in breast cancer cells. The human ER-α promoter (-4184/-1951) sequence was obtained by PCR from human genomic DNA and cloned into the pGL3-promoter vector (Promega, Wisconsin, USA). Mutagenesis of E2-boxes I and II in the human ER-α promoter was performed using a QuikChange Lightning Site-Directed Mutagenesis kit (Stratagene, Santa Clara, USA). Primer sequences are listed in Supplementary Data .
3
Cloning and Mutagenesis of Zeb1 Promoter
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The human complementary DNA (cDNA) fragment encoding the full-length Zeb1 sequence51 (link) was prepared by PCR and cloned into pLV-EF1-MCS-IRES-Bsd (Biosettia). The lentiviral-based vector pLV-H1-EF1α-puro (Biosettia) was used to express shRNAs in breast cancer cells and HUVECs. The human ZEB1 promoter (−1915/+132) sequences were obtained by PCR from human genomic DNA and cloned into the pGL3-basic vector (Promega). Mutagenesis of NRE-I and NRE-II in the human Zeb1 promoter was performed using a QuikChange® Lightning Site-Directed Mutagenesis kit (Stratagene). Primer sequences are listed in Supplementary Table 3 .
4
Cloning and Verification of WIP1, MKK3E, and MKK6A Plasmids
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The full-length CDS fragment of WIP1 was generated by polymerase chain reaction (PCR) and inserted into pLV-EF1-MCS-IRES-Bsd (Biosettia, USA). Single-strand oligos of shRNAs specific for WIP1 were synthesized, annealed into double-stranded oligos, and inserted into pLV-H1-EF1α-puro (Biosettia) following the guidance of the manufacturer’s protocol. DNA sequencing was then used to verify the plasmids. The human MKK3E and MKK6A plasmids were described previously.39 (link) Primer sequences are provided in the Supplementary Materials (Table S2 ).
5
Overexpression and knockdown of RBBP7 in tumor cells
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The complete coding sequence of the human RBBP7 gene was cloned into vector pLV-EF1-MCS-IRES-Bsd (Biosettia, San Diego, CA, USA). Lentiviruses were produced in 293T cells for the stable transfection of the cell lines, and an empty vector was used as the control. Tumor cells (1×105) were infected with 100 µl of lentivirus supernatant (1×1010 TU/ml). Forty-eight hour later, blasticidin (InvivoGen, San Diego, CA, USA) was added for selection.
Short interfering RNA (shRNA) sequences were designed using Biosettia's shRNA designer (http://biosettia.com/support/shrna-designer/ ). Three recommended sequences for RBBP7 were synthesized and cloned into the pLV-hU6-EF1α-puro vector (Biosettia). Scrambled sequences were used as controls. The lentiviruses were then produced in 293T cells. The most efficient of the three stable cell lines was used for the relevant assays.
Short interfering RNA (shRNA) sequences were designed using Biosettia's shRNA designer (
6
Molecular Cloning and Manipulation of ZEB1 and ATM
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The human complementary DNAs (cDNAs) fragment encoding the full-length ZEB1 sequence41 (link) was prepared by PCR and cloned into pLV-EF1-MCS-IRES-Bsd (Biosettia). The lentiviral-based vector pLV-H1-EF1α-puro (Biosettia) was used to express shRNAs in breast cancer cells. The human ATM promoter (−1534/+235) sequences were obtained by PCR from human genomic DNA and cloned into pGL3‑basic vector (Promega). Mutagenesis of the E2‑box in the human ATM promoter was performed using a QuikChange Site‑Directed Mutagenesis kit (Stratagene).
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