Low level of Hox1.3 gene expression does not preclude the use of promoterless vectors to generate a targeted gene disruption. off.
A variety of experimental approaches have been devised recently to mutate mammalian genes by homologous recombination. In this report, we describe the disruption of the Hox1.3 locus by using two of these approaches, namely, positive-negative selection and activation of a promoterless gene. Interestingly, we observe similarly high frequencies of targeted disruption with both procedures. The high frequency of targeted disruption with a promoterless vector was unexpected given the extremely low level of Hox1.3 expression in the embryonic stem cell line used for these studies. These data indicate that minimal expression of the target gene is required to enrich for homologous recombination events with promoterless vectors and thus enhance the promoterless gene approach as a general strategy to mutate mammalian genes by homologous recombination.
Jeannotte
L
L
Department of Genetics and Development, Columbia University College of Physicians & Surgeons, New York, New York 10032.
Ruiz
J C
JC
Robertson
E J
EJ
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005091:Exons; D015870:Gene Expression; D005801:Genes, Homeobox; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011995:Recombination, Genetic; D015183:Restriction Mapping
D009841:Oligonucleotides; D012313:RNA; D004247:DNA