Cloning and Mutagenesis of Zeb1 Promoter

The human complementary DNA (cDNA) fragment encoding the full-length Zeb1 sequence^{51 (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.

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Jiang H., Zhou C., Zhang Z., Wang Q., Wei H., Shi W., Li J., Wang Z., Ou Y., Wang W., Wang H., Zhang Q., Sun W., Sun P, & Yang S. (2020). Jagged1-Notch1-deployed tumor perivascular niche promotes breast cancer stem cell phenotype through Zeb1. Nature Communications, 11, 5129.

Publication 2020

pLV-EF1-MCS-IRES-Bsd pLV-H1-EF1α-puro pGL3-basic vector

Breast cancer Cdna Cells Human Human genomic Ires Mutagenesis Pgl3 Primer Promega Shrnas Site directed mutagenesis Vector

Corresponding Organization :

Other organizations : Nankai University, Capital Medical University, Tianjin First Center Hospital, Tianjin Medical University, Wake Forest University

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Variable analysis

independent variables

Preparation of human cDNA fragment encoding the full-length Zeb1 sequence by PCR and cloning into pLV-EF1-MCS-IRES-Bsd vector
Expression of shRNAs in breast cancer cells and HUVECs using the pLV-H1-EF1α-puro lentiviral-based vector
Mutagenesis of NRE-I and NRE-II in the human Zeb1 promoter using a QuikChange® Lightning Site-Directed Mutagenesis kit

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

No positive or negative controls were explicitly mentioned in the provided information.

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