Method of obtaining mutants defective in immunoglobulin mu transcription factors.
A method of obtaining mutants defective in the regulatory function of Ig mu gene expression was developed. Such mutants are useful for discovering the functions of transcription factors and isolating their genes, especially those of DNA-unbinding factors. Cells expressing Ig mu and Eco-GPT simultaneously under control of each mu enhancer were prepared as follows: myeloma X63Ag8.653 cells were transfected with pSV-V mu Me delta CH1 encoding a mu gene modified for cell surface mu expression without light chains, selected by neomycin resistance, transfected with Eco-GPT gene connected to the mu enhancer, and selected with mycophenolic acid in a medium containing xanthine. The mu(m)/Eco-GPT cells were mutated with ethane methyl sulfonate (EMS), and selected with toxin-conjugated anti-mu antibody, and then with 6-thioguanine. The mu(M)-/Eco-GPT- mutants obtained were fused with X63Ag8.653 cells. Fusion caused the mutant to recover mu(M) expression, suggesting that some trans-acting transcription factor other than the mu-encoding gene itself was probably mutated.
Shindo
T
T
Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Japan.
Ueda
H
H
Makishima
F
F
Nishimura
H
H
Suzuki
E
E
D016475:3T3 Cells; D000818:Animals; D002459:Cell Fusion; D002478:Cells, Cultured; D005434:Flow Cytometry; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D007075:Immunoglobulin M / Q000235:genetics*; D051379:Mice; D009154:Mutation / Q000502:physiology*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection
D007075:Immunoglobulin M; D014157:Transcription Factors; D007041:Hypoxanthine Phosphoribosyltransferase