Targeted gene disruption of the endogenous c-abl locus by homologous recombination with DNA encoding a selectable fusion protein.
We have introduced a substitution mutation into the c-abl locus of murine embryonic stem cells by homologous recombination between exogenously added DNA and the endogenous gene. Model constructs were initially generated that consisted of a promoterless selectable neomycin resistance marker inserted into the v-abl gene of the complete Abelson murine leukemia virus genome, designed to be expressed either as a fusion protein or by translational restart. Tests of these viral genomes for transmission of v-abl and neo markers showed more stable coexpression in a protein fusion construct. The neo fusion was subcloned from this v-abl construct into a promoterless c-abl fragment, and the resulting DNA was used to transform embryonic stem cells. Direct screening of genomic DNAs showed that a high proportion of drug-resistant clones arose from homologous recombination into the endogenous c-abl locus.
Schwartzberg
P L
PL
Department of Biochemistry and Molecular Biophysics, Columbia University, College of Physicians and Surgeons, New York, NY 10032.
Robertson
E J
EJ
Goff
S P
SP
D000818:Animals; D001483:Base Sequence; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics*; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000302:isolation & purification; D016315:Proto-Oncogene Proteins c-abl; D011519:Proto-Oncogenes; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D011995:Recombination, Genetic; D015183:Restriction Mapping
D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D011505:Protein-Tyrosine Kinases; D016315:Proto-Oncogene Proteins c-abl