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Homologous Recombination at c-fyn Locus of Mouse Embryonic Stem Cells with Use of Diphtheria Toxin A-Fragment Gene in Negative Selection
Takeshi Yagi, Yoji Ikawa, Keiichiro Yoshida, Yasuyo Shigetani, Naoki Takeda, Issei Mabuchi, Tadashi Yamamoto and Shinichi Aizawa
Proceedings of the National Academy of Sciences of the United States of America
Vol. 87, No. 24 (Dec., 1990), pp. 9918-9922
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/2356525
Page Count: 5
You can always find the topics here!Topics: Genes, Germ cells, Feeder cells, Cultured cells, Embryonic stem cells, Negative selection, Signals, L cells, Polymerase chain reaction, Exons
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In attempting to produce a mutant mouse with embryonic stem cells, the critical step is the efficient isolation of homologous recombinants; the frequency of the homologous recombination is usually low and the potency of the cells to differentiate into germ cells is unstable in culture. Here, we report an efficacious method for such isolation in which the diphtheria toxin A-fragment gene is used to negatively select nonhomologous recombinants. In contrast to the use of the herpes simplex virus thymidine kinase gene, the selection can be made singly by the neomycin analog G418 without using a drug such as ganciclovir, a nucleoside analog. At the c-fyn locus, the diphtheria-toxin negative selection enriched the recombinants about 10-fold, and half of the cells integrating with the neomycin phosphotransferase gene were homologous recombinants.
Proceedings of the National Academy of Sciences of the United States of America © 1990 National Academy of Sciences