This knock-out results in the complete ablation of the eosinophil lineage. This mutant may be useful for in vivo studies of eosinophil function and eosinophil-related pathologies, including asthma and pulmonary physiology.
Stuart H Orkin, Harvard University
Homozygous females and hemizygous males for the targeted mutation are viable, fertile, and normal in size. This mutation results in the complete ablation of the eosinophil lineage, even under conditions that normally stimulate eosinophil development, without affecting the development of other GATA-1 dependent lineages (erythroid, megakaryocytic, and mast cells). Expression of the endogenous gene is observed in erythroid and bone marrow cells. This mutant may be useful for in vivo studies of eosinophil function and eosinophil-related pathologies, including asthma and pulmonary physiology.
A targeting vector was designed to replace the double GATA-site 21 bp upstream of the first hematopoietic exon of the X-linked endogenous gene. This vector, containing a loxP-flanked phosphoglycerate kinase promoter driven neomycin resistance gene (PGK-neo), was electroporated into 129S1/Sv derived CJ-7 embryonic stem (ES) cells. Correctly targeted ES cells were then transiently transfected with a cre recombinase vector to remove the PGK-neo resulting in a mutant allele where the double GATA-site is replaced with a single loxP site. These cells were injected into C57BL/6 blastocysts and the resulting chimeric males were backcrossed to C57BL/6 females. Resultant heterozygotes were backcrossed to BALB/c for eight generations before being made homozygous/hemizygous.
|Allele Name||targeted mutation 6, Stuart Orkin|
|Allele Type||Targeted (Null/Knockout)|
|Allele Synonym(s)||dblGATA1; deltadbl GATA; deltadblGATA; Eos-; Gata1tm5Sho|
|Gene Symbol and Name||Gata1, GATA binding protein 1|
|Strain of Origin||129S1/Sv-Oca2+ Tyr+ Kitl+|
|Molecular Note||A high-affinity, palindromic "double" GATA protein binding site in the Gata1 promoter presumed to mediate positive Gata1 autoregulation was replaced by a floxed Pgk-neo cassette; transient Cre recombinase expression in ES cells left a single loxP site flanked by two Not1 sites. The 21-bp deleted segment comprised nucleotides -691 through -671 upstream of the last nucleotide of the first hematopoietically expressed exon of Gata1.|
|Mutations Made By|| |
Stuart Orkin, Harvard University
When maintaining a live colony, this colony is maintained as homozygotes.
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