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DNA‐mediated transformation of mammalian cells in culture. Increased transforming efficiency following sonication
Author(s) -
Roosa Robert A.,
Bailey Ellen
Publication year - 1970
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1040750202
Subject(s) - sonication , dna , transformation (genetics) , microbiology and biotechnology , rnase p , biology , transformation efficiency , chemistry , biochemistry , rna , chromatography , gene , agrobacterium
A series of 100 experiments was completed to determine if DNA is capable of transforming the genotype of a murine lymphoma (P388) in cell culture. The test system was concerned with the transformation of cells from 8‐azaguanine (AZG) sensitivity to resistance. By the use of this marker, it was determined that transformation by DNA did occur, and that the efficiency of transformation was greatly increased by sonication of the DNA. A statistical analysis of 100 experiments demonstrated that the increase in the number of resistant cells after treatment with sonicated resistant DNA (R‐DNA) was statistically significant (χ 2 > 4.25, 0.05 > p > 0.02) in 66% of the experiments. DNA from sensitive parental cells and DNA from other sources produced no effect while DNase and UV treatment abrogated effective transformation by either sonicated or nonsonicated R‐DNA. RNase was without effect. Sucrose gradient analysis of sonicated and nonsonicated R‐DNA demonstrated that the peaks which correspond to the highest specific transforming activity are not altered by sonication and do not coincide with the OD 260 peaks, in spite of the fact that sonication shifted the peak of maximum OD 260 to a slower sedimenting region of the gradient. The major portion, however, of the transforming material did shift after sonication to the slower sedimenting region of the gradient and did coincide with the OD 260 peak. The hereditary stability of the transformed cells was established by cloning a representative number of transformants, growing them in the absence of AZG for an extended period and then testing their ability to grow in graded concentrations of AZG. In addition, DNA extracted from transformants successfully transformed sensitive cells.