Efficient modification of human chromosomal alleles using recombination-proficient chicken/human microcell hybrids.
Targeted modification of human chromosomal alleles by homologous recombination is a powerful approach to study gene function, but gene targeting in mammalian cells is an inefficient process. In contrast, gene targeting in a chicken pre-B cell line, DT40, is highly efficient. We have transferred human chromosome 11 into DT40 cells by microcell fusion, and find that the resulting hybrids are recombination-proficient. In these cells, targeting efficiencies into the chicken ovalbumin locus were > 90% and into the human beta-globin and Ha-ras loci were 10-15%. These modified human chromosomes can be transferred subsequently to mammalian cells for functional tests. This chromosome shuttle system allows for the efficient homologous modification of human chromosomal genes, and for subsequent phenotypic analyses of the modified alleles in different mammalian cell types.
Dieken
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ES
Division of Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, USA.
Epner
E M
EM
Fiering
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S
Fournier
R E
RE
Groudine
M
M
D000483:Alleles; D000818:Animals; D001402:B-Lymphocytes; D001483:Base Sequence; D002459:Cell Fusion; D002460:Cell Line; D002645:Chickens; D002880:Chromosomes, Human, Pair 11; D018390:Gene Targeting / Q000379:methods*; D011905:Genes, ras / Q000235:genetics; D005914:Globins / Q000235:genetics; D006801:Humans; D006822:Hybrid Cells; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D011995:Recombination, Genetic / Q000235:genetics*; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D005914:Globins; D010047:Ovalbumin