Homologous recombination at c-fyn locus of mouse embryonic stem cells with use of diphtheria toxin A-fragment gene in negative selection.
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.
Yagi
T
T
Laboratory of Molecular Oncology, Tsukuba Life Science Center, Physical and Chemical Research Institute (RIKEN), Ibaraki, Japan.
Ikawa
Y
Y
Yoshida
K
K
Shigetani
Y
Y
Takeda
N
N
Mabuchi
I
I
Yamamoto
T
T
Aizawa
S
S
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004167:Diphtheria Toxin / Q000235:genetics*; D004622:Embryo, Mammalian; D005260:Female; D015723:Gene Library; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D051076:Proto-Oncogene Proteins c-fyn; D011519:Proto-Oncogenes; D015183:Restriction Mapping; D012641:Selection, Genetic
D004167:Diphtheria Toxin; D015345:Oligonucleotide Probes; D011518:Proto-Oncogene Proteins; D004247:DNA; D011505:Protein-Tyrosine Kinases; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn