pHyPer-nuc is a mammalian expression vector encoding nuclear-targeted HyPer (see reporter description). HyPer codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Three copies of the nuclear localization signal (NLS) fused to the HyPer C-terminus provide for efficient translocation of HyPer to the nuclei of mammalian cells [Fischer-Fantuzzi and Vesco, 1988].

Ratiometric imaging of HyPer response to H2O2 in HeLa cells nuclei.

HeLa cells expressing HyPer-nuc were plated to glass bottom dishes and exposed to 180 μM H2O2. Images were acquired by Leica AF 6000 LX with 0.5 Hz frequency by sequential illumination of cells via CFP/YFP (excitation/emission) and YFP/YFP filters. Resulting images were obtained by dividing of YFP/YFP images to CFP/YFP images followed by pseudo coloring.

pHyPer-nuc vector can be used as a source of HyPer-NLS hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice. Alternatively, HyPer-NLS coding sequence can be amplified by PCR.

Note: The plasmid DNA was isolated from dam+-methylated E. coli. Therefore some restriction sites are blocked by methylation. If you wish to digest the vector using such sites you will need to transform the vector into a dam- host and make fresh DNA.

The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation signals (SV40 poly A) direct proper processing of the 3'-end of the reporter mRNA.

SV40 early promoter (PSV40) provides neomycin resistance gene (Neor) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kanr) in E. coli. Kanr/Neor gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.


Expression in mammalian cells

pHyPer-nuc vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive HyPer expression in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].


Propagation in E. coli

Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin (30 μg/ml) to E. coli hosts. Copy number in E. coli is about 500.