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Title: Method for rapid isolation of sensitive mutants

Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned.
Inventors:
 [1]
  1. (Los Alamos, NM)
Issue Date:
OSTI Identifier:
871073
Assignee:
United States of America as represented by United States (Washington, DC) LANL
Patent Number(s):
US 5652098
Contract Number:
W-7405-ENG-36
Research Org:
Los Alamos National Laboratory (LANL), Los Alamos, NM
Country of Publication:
United States
Language:
English
Subject:
method; rapid; isolation; sensitive; mutants; mammalian; cell; rapidly; isolated; flow; cytometry; population; clonal; spheroids; established; contain; normal; mutant; cells; naturally; occurring; arise; mutagenized; sorted; size; obtain; uniform; exposed; dna-damaging; agent; investigated; placed; growth; medium; form; third; comprising; increased; resistant; substantially; formed; differentiate; treated; recover; viable; line; cloned; naturally occurring; flow cytometry; uniform size; mammalian cells; growth medium; cell line; mutant cells; flow sorted; /435/

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