ABSTRACT
The dominant selective markers that we and others have constructed for the selection of cells which stably acquired a foreign DNA, are briefly described. The possibility of obtaining the cointegration of several genes, and of inducing their amplification in the recipient cell, is discussed.
Subject(s)
Genetic Markers , Transfection/methods , Adenosine Deaminase/genetics , Alcohol Oxidoreductases/genetics , Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Cells, Cultured , Dihydroorotase/genetics , Kanamycin Kinase , Multienzyme Complexes/genetics , Pentosyltransferases/genetics , Phosphotransferases/genetics , Recombinant Proteins/biosynthesis , Tetrahydrofolate Dehydrogenase/geneticsSubject(s)
Viral Vaccines , Yellow fever virus/immunology , Drug Stability , Freeze Drying , Hot TemperatureABSTRACT
We studied the cotransfer and cointegration of several genes transfected into four cell lines of primate origin. Mouse thymidine-kinase-negative LM cells, which had been extensively studied previously, were used as a reference. We found that in monkey kidney Vero cells, on average between 3.5 and 6.0 kb of plasmid sequences was integrated per clone, while in the murine LM cell line, 9-186 kb of exogenous DNA was integrated per clone. Transformed Vero clones which had integrated more than 6 kb of DNA did not integrate larger DNA fragments in a second transformation assay than had the parental Vero cells. We found that the efficiency of gene cointegration is similar in Vero, HeLa and GM4312A cells, the latter being deficient in the repair of UV-induced damage. The human hepatocarcinoma Hep G2 cells integrated on the average 2 kb more exogenous DNA than the three other primate cell lines, which resulted in a 4-5 times higher efficiency of gene cointegration. Plasmid penetration and persistence in a free state between 24 h and two weeks after transfection was similar in Vero and LM cells. No major post-integration DNA rearrangement could be demonstrated after the isolation of Vero clones. These observations correlate the low efficiency of gene cointegration in some primate cell lines with a genomic recombination step or with rearrangements taking place during early cell divisions following integration.
Subject(s)
Primates/genetics , Rodentia/genetics , Transfection , Animals , Cell Line , DNA, Recombinant , Gene Expression Regulation , Hepatitis B Surface Antigens/immunology , Humans , Kanamycin Kinase , Phosphotransferases/genetics , Recombination, Genetic , Species Specificity , Transformation, Genetic , Vero CellsABSTRACT
The infectious units in native and alkalidenatured preparations of DNA of herpes simplex virus were characterized with respect to their sensitivity to Neurospora crassa endonuclease, their sedimentation properties in high-salt, neutral sucrose gradients, and their sensitivity to hydrodynamic shearing forces. Infectious molecules in native preparations were resistant to N. crassa endonuclease, sedimented at 56 S, and were highly sensitive to shearing forces. After alkaline denaturation, infectious molecules became sensitive to the N. crassa enzyme, sedimented at 200 S, and were relatively resistant to shear. We conclude that both intact duplex molecules ([unk]100 x 10(6) daltons) and intact single strands ([unk]50 x 10(6) daltons) are capable of initiating productive infection.
