DHFR Coamplification of t-PA in DHFR⁺Bovine Endothelial Cells:In VitroCharacterization of the Purified Serine Protease

Abstract

High-level expression of human tissue-type plasminogen activator was accomplished in endothelial cells by a novel approach to dihydrofolate reductase (DHFR) coamplification in DHFR+ cells. A tripartite mammalian expression vector coding for DHFR, neomycin phosphotransferase, and the t-PA gene was introduced into bovine endothelial cells by transfection and selection for G418 resistance. Upon methotrexate selection of these transformants, we obtained endothelial cells that had amplified the plasmid-encoded DHFR and t-PA genes. As a result, cell lines were isolated that efficiently produced t-PA (greater than 4 pg/cell.day). This t-PA was purified and compared with recombinant t-PA produced in Chinese hamster ovary cells. These two t-PA samples differed in carbohydrate composition, and amounts of 530 and 527 amino acid forms but had similar in vitro activity.

Induction of sensitivity to the cytotoxic action of tumor necrosis factor alpha by adenovirus E1A is independent of transformation and transcriptional activation

“…RSVtatDB5 also contains the simian virus 40 (SV40) origin of replication for use in COS-1 cells (23 stream from the Rous sarcoma virus promoter, followed by the bovine growth hormone polyadenylation signal sequence. This plasmid also contains the neomycin resistance gene transcribed from the mouse major ,-globin promoter (13). Cell lines.…”

PC12 Cells Overexpressing Tissue Plasminogen Activator Regenerate Neurites to a Greater Extent and Migrate Faster than Control Cells in Complex Extracellular Matrix

“…pRSV-tPA ( Fig . 1) was generated similarly by digesting pRldn (Conners et al ., 1988) with HindIII, followed by blunt-end ligation of the full-length tPA cDNA into this blunted site . Ligation sites, reading frames, and orientation of tPA cDNA inserts were confirmed by nucleotide sequencing .…”

Structural and biological properties of human recombinant myeloperoxidase produced by Chinese hamster ovary cell lines

“…Plasmids pMP062 (Johnson et al, 1987) and pTDN (Connors et al, 1988) have been described earlier. Plasmid pTDN PC2, provided by Dr. Reff, is a derivative of pTDN which is used only as an intermediate vehicle in plasmid construction.…”

“…Map ofpNIV2703,2705 and 2706. The mammalian expression vector has been described before (Connors et al, 1988). The three cassettes direct the expression of a dihydrofolate-reductase-(DHFR)-selectable marker, recMPO and a neomycin-resistance (NeoR) selectable marker.…”