Transfection techniques for producing recombinant baculoviruses.

The production of recombinant baculoviruses usually employs cotransfection of insect tissue-culture cells with viral and transfer-plasmid DNAs. The preparation and storage of viral and plasmid DNAs suitable for optimal transfection of insect cells are discussed. Electroporation, calcium-phosphate, and lipofection transfection techniques are presented with a discussion of their relative advantages. The rates of recombinant virus formation are compared using viral infection/plasmid transfection protocols versus cotransfection of cells with transfer-plasmid and viral DNAs.

Baculovirus expression of alkaline phosphatase as a reporter gene for evaluation of production, glycosylation and secretion.

We have devised a simple and efficient baculovirus expression vector system to evaluate insect tissue culture cells for their capacity to express, glycosylate and secrete foreign proteins. A truncated placental alkaline phosphatase (SEAP) gene was inserted into the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome under the transcriptional control of the polyhedrin gene promoter. Production levels, glycosylation, and secretion of the recombinant protein were examined in Trichoplusia ni (BTI-TN-5B1-4) and Spodoptera frugiperda (Sf9) cell lines. The assay for SEAP activity, which is fast, inexpensive, and quantitative to concentrations of 20 picograms per milliliter, was used to assess cell-associated and secreted SEAP activity. The proportion of SEAP which is modified with N-linked oligosaccharide can also be determined due to the difference in mobilities during SDS-PAGE between the glycosylated and nonglycosylated forms of the protein.

Microtiter plate assays for the measurement of phage adsorption and infection in Lactococcus and Enterococcus.

Three easy and rapid microtiter plate assays for determining phage sensitivity of lactococci and enterococci have been developed. In the microlysis assay, the degree of sensitivity was measured on the basis of the ability of the bacterial cells to grow in the presence of various concentrations of phage and to effect a color change of an acid-base indicator as a result of acid production. Two assays that specifically measure phage adsorption to bacterial cells have been developed on the basis of the enzyme-linked immunosorbent assay (ELISA) technique. In the direct phage adsorption ELISA, adsorption of phage particles to cells immobilized onto microtiter plate wells was measured using specific anti-phage antibody. In the competitive phage adsorption ELISA, phage adsorption was assayed by allowing phage to compete with specific antibody binding to the bacterial cell surface. All three assays were quantifiable photometrically.

Mutants of Enterococcus faecalis deficient as recipients in mating with donors carrying pheromone-inducible plasmids.

From Enterococcus faecalis cells containing random chromosomal insertions of Tn916, strains resistant to a lytic phage were selected and tested for conjugal mating ability. The phage-resistant strains all showed decreased recipient ability (Con-) in broth matings with donors carrying pheromone-inducible plasmids. These strains were normal with respect to donor ability in broth matings and recipient ability in filter matings. The data suggest that the mutants are deficient in the binding substance receptor for the pheromone-induced donor aggregation substance. These mutants contained multiple insertions of Tn916, and none of the individual insertions from the mutant strains were capable of generating the phenotype. Analysis of cell envelope lipoteichoic acids and protein revealed changes in both associated with the Con- phenotype.

Modulation of macrophage fibronectin secretion by LPS.

The secretion of fibronectin (Fn) by rat peritoneal macrophages was found to be down-regulated by LPS. A sensitive ELISA was used to quantitate both substrate-attached and supernatant Fn. Thioglycollate-elicited peritoneal exudate cells were observed to release a considerable amount of Fn during the adherence procedure for macrophage purification. After this procedure, macrophage Fn levels peaked within 2 hr and then declined steadily to baseline levels by 96 hr. Fn release by exudate cells during adherence purification was less affected by cycloheximide treatment than was subsequent Fn secretion by purified macrophages. Macrophages elicited with thioglycollate and P. acnes displayed enhanced Fn secretory activity when compared with resident unstimulated cells. Exposure to lipopolysaccharide (LPS) suppressed the levels of immunoreactive Fn in supernatants of elicited cells. This inhibition was shown to be dose-dependent and most significant after 24 hr of incubation. The inclusion of polymyxin B in the culture medium did not reverse the LPS-induced inhibition of Fn production but did prevent LPS stimulation of interleukin-1 secretion in the macrophage cultures. These observations demonstrate that Fn secretion by macrophages is regulated according to the functional state of the cell.