Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases
A simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients has been developed that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39 degrees C. Hybrids were exposed to very high doses of gamma-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39 degrees C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39 degrees C to reduce the amount of human DNA even further. Using this procedure, Chinese hamster cell lines have been constructed that express the human genes encoding either asparaginyl- or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure.
- Research Organization:
- Univ. of California, Irvine, CA (United States)
- OSTI ID:
- 6748647
- Journal Information:
- Mol. Cell. Biol.; (United States), Vol. 3:5
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
CHROMOSOMES
GENETIC RADIATION EFFECTS
FIBROBLASTS
HYBRIDIZATION
LEUKOCYTES
DNA-CLONING
GAMMA RADIATION
GENE RECOMBINATION
HAMSTERS
LIGASES
MAN
MUTANTS
TRANSFER RNA
ANIMAL CELLS
ANIMALS
BIOLOGICAL EFFECTS
BIOLOGICAL MATERIALS
BIOLOGICAL RADIATION EFFECTS
BLOOD
BLOOD CELLS
BODY FLUIDS
CLONING
CONNECTIVE TISSUE CELLS
ELECTROMAGNETIC RADIATION
ENZYMES
GENETIC EFFECTS
IONIZING RADIATIONS
MAMMALS
MATERIALS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PRIMATES
RADIATION EFFECTS
RADIATIONS
RNA
RODENTS
SOMATIC CELLS
VERTEBRATES
560121* - Radiation Effects on Cells- External Source- (-1987)
550400 - Genetics