Mammalian cell lines can be efficiently established in vitro upon expression of the SV40 large T antigen driven by a promoter sequence derived from the human vimentin gene

Abstract

The aim of this study was to investigate a new method to enhance the efficiency to create mammalian cell lines. Cell immortalization was achieved by intranuclear microinjection of a recombinant DNA construct composed of a constitutive promoter controlling the genes encoding immortalizing proteins; the sequences coding for the large T and small t antigens were fused downstream of regulatory elements from the vimentin gene, the activation of which characterizes the vast majority of cells growing in vitro. Data show that the efficiency of the immortalizing procedures using the SV40 early genes could be enhanced by the control elements derived from the human vimentin (HuVim) 5' sequences that contained nucleotides -878 to +93 from the CAP site. This HuVim 830-T/t recombinant was used to create cell lines from numerous primary cultures of different origins: rabbit, porcine and human endothelial cells, rabbit and bovine epithelial cells. A set of large T-expressing cells was derived, and these cells retained characteristics of differential cells: binding of Ulex europaeus lectin and synthesis of Factor VIII for human endothelial cells; network of cytokeratin for bovine oviductal cells and rabbit mammary cells.

Cultured Peripheral Neuroglial Cells Are Highly Permissive to Sheep Prion Infection

“…The transgenic animals used to derive stable mouse cell clones expressing ovine PrP were obtained by crossing the following two parental lines: the tg301 ϩ/Ϫ line, which expresses the Val136-Arg154-Gln171 high-susceptibility allele of ovine PrP from a bacterial artificial chromosome construct (125 kb) under an endogenous PrP-null background (tgOv) (58), and a line which carries a transgene specifying the simian virus 40 (SV40) T antigen (Tag) under control of the vimentin promoter (52), which was introduced on the same mouse PrP-null background (PrP 0/0 mouse Zürich I) (11). DRG primary cultures and stable cell clones.…”

Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules

“…The plasmid pHu Vim 830-T/t contained DNA encoding the simian virus 40 large tumor antigen under the control of the human vimentin promoter (15). For the transfection assays, 2 g of plasmid were incubated with 20 g of mAb in 20-30 l of PBS for 15 min at room temperature, and the volume was adjusted with complete culture medium to a final volume of 0.5 ml.…”

Immortalized Human Brain Endothelial Cell Line HCMEC/D3 as a Model of the Blood-Brain Barrier Facilitates In Vitro Studies of Central Nervous System Infection by Cryptococcus neoformans

“…The alternative, obtaining primary HBMEC from discarded brain tissues, is also undesirable since the process is labor-intensive and introduces variability from batch to batch (4,8). To facilitate the study of the BBB in vitro, researchers have tried to develop human brain endothelial cell lines that retain critical features of primary cells, such as the expression of endothelial cell markers, transporters, and tight junctional proteins (1,15,23,25,27,29,30,33,34,36). The recent development of one particular line of immortalized human brain endothelial cells (HCMEC/D3) that recapitulates many of the key characteristics of primary brain endothelial cells without the need to coculture with glial cells is proving to be a promising cell line for in vitro studies of the BBB (36).…”