Homologous recombination at c-fyn locus of mouse embryonic stem cells with use of diphtheria toxin A-fragment gene in negative selection.

• DOI: 10.1073/pnas.87.24.9918
• Publication Date: 2006-05-31T11:32:52Z
• Authors: T Yagi; Y Ikawa; K Yoshida; Y Shigetani; N Takeda; I Mabuchi; T Yamamoto; S Aizawa
• Keywords: 0301 basic medicine; Mice, Inbred BALB C; 0303 health sciences; Base Sequence; Genetic Vectors; Molecular Sequence Data; DNA; Protein-Tyrosine Kinases; Embryo, Mammalian; Hematopoietic Stem Cells; Proto-Oncogene Proteins c-fyn; Cell Line; 3. Good health; Mice, Inbred C57BL; Blotting, Southern; Mice; 03 medical and health sciences; Proto-Oncogene Proteins; Proto-Oncogenes; Animals; Diphtheria Toxin; Female; Oligonucleotide Probes; Gene Library

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.