Pcdna3.1 nt gfp topo vector

Site-directed mutagenesis was performed following a previously described technique [58 (link)] with slight modifications. Namely, to introduce a nucleotide substitution into the extended DNA fragments corresponding to VP2 and VP3 coding sequences, a two-step PCR was carried out. The first step included the amplification of two DNA fragments overlapping by 25 nucleotides, with the region of overlap corresponding to the DNA region into which the mutation should be introduced. The reverse primer for the upstream fragment and the forward primer for the downstream fragment used in the first step contained the desired nucleotide substitution and the other two primers corresponded to the ends of the entire DNA fragment being mutated. In the second step, the extended DNA fragment was amplified using the overlapping DNA fragments obtained at the previous step as a template and the pair of primers flanking the entire mutated DNA fragment. Tersus Plus PCR kit (Evrogen, Moscow, Russia), containing a mixture of high fidelity polymerases, was used to perform PCR. The obtained extended DNA fragments were cloned into the pcDNA3.1/NT-GFP-TOPO vector (Invitrogen, USA) as described above. The mutagenesis and vector construction accuracy, as well as the absence of PCR-introduced errors, were all verified by Sanger sequencing.

pcDNA3.1-v5-hYSK1 and deletion fragments of hYSK1 were generated by PCR using the human cDNA clone-hYSK1 (Origene Technologies, MD, USA) as a template. The PCR product was purified, digested with EcoRI/XhoI, and cloned into the EcoRI/XhoI sites of pcDNA3.1-v5-HisA (Invitrogen, MA, USA). For TOPO-GFP-hYSK1, the PCR product was inserted into the pcDNA3.1-NT-GFP-TOPO vector (Invitrogen, MA, USA). The GST-p16^INK4a and GST-deletion fragments of p16^INK4a were inserted in-frame into the BamHI/XhoI site of the pGEX-5X-1 vector (Amersham Biosciences, PA, USA). For pDsRed-p16^INK4a, the PCR product was cloned into the ApeI site of the pDsRed vector (Clontech Laboratories, CA, USA). HA-SP-1 was cloned into the EcoRI/XhoI site of the pCMV-HA and pCMV-Myc vectors (Clontech Laboratories, CA, USA). The shRNA-hYSK1 plasmid was constructed into the pSilencer 4.1-CMV-hyg vector (Ambion, NY, USA). The pGL2-p16-luc vector was a gift from Dr. Gordon Peters (Cancer Research UK, London) and the pGL2-MMP-2-luc vector was a gift from Dr. Etty N. Benveniste (The University of Alabama at Birmingham, Birmingham, AL). Various expression vectors were amplified in E. coli XL1-blue or BL21 cells and plasmids were purified using a Qiagen midi kit (Qiagen, Hilden, Germany). The DNA sequences of all plasmids were confirmed by sequencing (Dye Terminator ABI Type Seq., Bionex, NJ, USA)

A pCMS-EGFP vector containing the wild-type (WT) KCNJ2 cDNA was kindly provided by Prof. Minoru Horie (Shiga University of Medical Science, Japan; Haruna et al., 2007 (link)). The c.173T>C (p.Phe58Ser) mutation was introduced by site-directed mutagenesis using the Quick Change II XL kit (Stratagene, La Jolla, CA, USA). The cDNA was completely resequenced after mutagenesis. WT or mutant cDNA was sub-cloned into a pcDNA3.1-NT-GFP-TOPO vector (Invitrogen), so that the GFP protein was fused at the N-terminus of the Kir2.1 channel. Plasmids were purified using QIAGEN Plasmid Maxiprep kit (QIAGEN, Hilden, Germany) following the suggested protocol. All constructs were verified by sequence analysis.