Physical and biological properties of cationic triesters of phosphatidylcholine.

The properties of a new class of phospholipids, alkyl phosphocholine triesters, are described. These compounds were prepared from phosphatidylcholines through substitution of the phosphate oxygen by reaction with alkyl trifluoromethylsulfonates. Their unusual behavior is ascribed to their net positive charge and absence of intermolecular hydrogen bonding. The O-ethyl, unsaturated derivatives hydrated to generate large, unilamellar liposomes. The phase transition temperature of the saturated derivatives is very similar to that of the precursor phosphatidylcholine and quite insensitive to ionic strength. The dissociation of single molecules from bilayers is unusually facile, as revealed by the surface activity of aqueous liposome dispersions. Vesicles of cationic phospholipids fused with vesicles of anionic lipids. Liquid crystalline cationic phospholipids such as 1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine triflate formed normal lipid bilayers in aqueous phases that interacted with short, linear DNA and supercoiled plasmid DNA to form a sandwich-structured complex in which bilayers were separated by strands of DNA. DNA in a 1:1 (mol) complex with cationic lipid was shielded from the aqueous phase, but was released by neutralizing the cationic charge with anionic lipid. DNA-lipid complexes transfected DNA into cells very effectively. Transfection efficiency depended upon the form of the lipid dispersion used to generate DNA-lipid complexes; in the case of the O-ethyl derivative described here, large vesicle preparations in the liquid crystalline phase were most effective.

Trophoblast-specific transcription from the mouse placental lactogen-I gene promoter.

We have isolated the gene encoding mouse placental lactogen-I and characterized the promoter region of this gene by transient and stable transfection. Promoter sequences extending 274 basepairs (bp) up-stream from the start site of transcription contain all of the elements necessary for maximal expression upon transient transfection into the rat choriocarcinoma Rcho-1 cell line; these Rcho-1 cultures contain both proliferative trophoblast stem cells and terminally differentiated trophoblast giant cells. In stably transfected cell lines, expression from this promoter increases as the percentage of differentiated cells in the culture increases. In contrast to these results in trophoblast cells, the 274-bp promoter as well as a promoter region extending 2700 bp up-stream of the transcriptional start site are unable to drive transcription in a variety of other cell types. Mutational and protein binding analyses indicate that two AP-1 sites are required for maximal expression in Rcho-1 cells, and that the composition of the AP-1 transcription factor may vary as differentiation in the cell culture increases. In addition to these two AP-1 sites, at least one other element appears to be critical for promoter activity in trophoblast cells.

Placental-specific expression from the mouse placental lactogen II gene promoter.

The gene for mouse placental lactogen II (mPL-II) has been isolated and characterized. This gene contains five exons, with a transcription start site 59 nucleotides upstream of the translation initiation ATG. Introduction of a DNA construct containing 2.7 kilobases of sequence upstream of the mPL-II transcription initiation site directed the synthesis of a linked coding region for the simian virus 40 large and small tumor antigens in placental trophoblast giant cells of transgenic mice. The pattern of simian virus 40 transgene expression in the placenta was indistinguishable from that of the endogenous mPL-II gene. In contrast, the first 569 base pairs upstream of the transcription start site proved insufficient to direct placental expression. Thus, one or more elements required for placental trophoblast giant cell expression have been localized to a region between -2700 and -569 of the mPL-II gene.