pPhi-Yellow-mito is a mammalian expression vector intended for yellow fluorescent labeling of mitochondria in living cells. The vector encodes yellow fluorescent protein PhiYFP (see reporter description) fused to mitochondrial targeting sequence (MTS) derived from the subunit VIII of human cytochrome C oxidase [Rizzuto et al., 1989; Rizzuto et al., 1995]. MTS is fused to the PhiYFP N-terminus.

Stably transfected PtK rat kangaroo cells expressing mitochondria-targeted PhiYFP.

Image was kindly provided by Dr. Christian Petzelt (Marinpharm).

PhiYFP codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996].

pPhi-Yellow-mito vector can be used as a source of PhiYFP-MTS hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice.

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 (P_{CMV 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 (P_SV40) provides neomycin resistance gene (Neo^r) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kan^r) in E. coli. Kan^r/Neo^r gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.

Expression in mammalian cells

pPhi-Yellow-mito vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the PhiYFP-MTS fusion 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.