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Blood, Vol. 107, Issue 9, 3527-3530, May 1, 2006
Phosphorylation of Gata1 at serine residues 72, 142, and 310 is not essential for hematopoiesis in vivo
Blood Rooke and Orkin
107: 3527
Supplemental materials for: Rooke and Orkin
Files in this Data Supplement:
- Figure S1. Gene targeting approach used to generate the Gata1S310A and Gata13SA knock-in alleles (JPG, 72.2 KB)
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The wild-type allele was targeted with a vector containing a Gata1 genomic fragment spanning exons 2-6 with point mutations introduced to encode a serine to alanine replacement at residue 310 alone (A) or together with residues 72 and 142 (B). The vector contained a neomycin resistance gene (neo) flanked by loxP sites and a thymidine kinase negative selection cassette as described previously13. The 5’ homologous arm of the Gata13SA construct was extended by addition of an XbaI-EagI genomic fragment. Point mutations encoding serine to alanine replacements were introduced by use of the Quick Change XL Site-Directed Mutagenesis kit (Promega). Correctly targeted clones were identified by Southern blot and PCR strategies13,14. Mutations encoding serine to alanine substitutions were confirmed by sequencing PCR products spanning the relevant region. Neo was excised from the Gata13SA allele in vitro as described14. Mice were generated by the injection of targeted ES cells into C57BL/6 blastocysts to generate chimeras that were crossed with C57BL/6 females. Neo was excised from the Gata1S310A allele by crossing the knock-in mice with a Gata1-cre transgenic mouse. Each Gata1 knock-in strain was back-crossed to C57BL/6 for several generations. Routine genotyping was performed as described14. Serine to alanine substitutions were confirmed in a sample from each generation.
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