Fast site directed mutagenesis kit

The Full-length fragment of NDRG1 was amplified from a iSLK.RGB cDNA library, inserted into Streptomycin-Flag-tagged pCDH and HA-tagged pCMV vectors. The KSHV ORF44, RTA expression plasmids were previously described [22 (link),76 (link)]. Mammalian expression plasmids for ORF44 and NDRG1 truncations were constructed by standard molecular biology techniques (see the schematics in Fig 2A, 2C and 2E). The ORF44 mutant plasmids were generated by using the pCMV-HA-ORF44 plasmid as a template following the manufacturer’s protocol of the Fast Site-Directed Mutagenesis Kit (TIANGEN) (see the schematics in Fig 7A). The luciferase reporter plasmid pGL3-Basic-pNDRG1 was constructed by cloning the promoter regions of NDRG1 (-2000 to -1 bp) from the iSLK.RGB genomic library into the pGL3-Basic vector. The reporter plasmid pGL3-Basic-pNDRG1-ΔRBP-Jκ was constructed by using the pGL3-Basic-pNDRG1 plasmid as a template and the Fast Site-Directed Mutagenesis Kit (TIANGEN) (see the schematics in Fig 3C). All the primers used for gene amplification are listed in S2 Table.

To predict miR-212/132-binding sites in the 3′ UTR of FBW7, firefly luciferase reporter vectors were constructed with the psiCHECK2 plasmid and FBW7 3′ UTR. A mutation at the nucleotide position of the miRNA seed sequence in the FBW7 3′ UTR was generated using a Fast Site-Directed Mutagenesis Kit (Tiangen) in accordance with the manufacturer’s instructions. Lipofectamine 3000 was used to transfect HEK293T cells with a mixture of the firefly luciferase reporter plasmid, miRNA precursor or control precursor, and Renilla reniformis luciferase-encoding plasmid. Cells transfected without precursors served as controls for normalization.
To detect promoter activity of miR-212/132, serial mutated fragments of the promoter region were inserted into pGL3.0-Luc. The plasmids and Renilla luciferase expression vector (pRL-40) were cotransfected into HEK293T cells. To examine the effect of transcription factors on promoter activity, PDX1 or NGN3 expression plasmids were mixed with pRL-40 and promoter luciferase reporter constructs with normal or mutant binding sites and cotransfected into HEK293T cells. Luciferase activity was measured at 48 h post-transfection using a dual-luciferase assay system. All detections were repeated independently at least three times.

Bioinformatics analysis of the regions with high transcriptional activity revealed two transcription factors EGR1 and SP1 with high scores. To verify whether these two transcription factors have significant effects on transcription activity, transcription factor binding sites were mutated. According to the primer design requirements of the Fast Site-Directed Mutagenesis Kit (Tiangen, Beijing), the transcription factor point mutation primers (Table 2) were designed using Oligo7. The mutation plasmid BT1 (mutation of EGR1 binding site) and BT2 (mutation of SP1 binding site) were constructed using the B5 fragment as a template, respectively. The EGR1 overexpression plasmid was constructed by Suzhou Jinglun Biotechnology Co., Ltd., Suzhou, China.