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Blood, Vol. 105, Issue 7, 2741-2748, April 1, 2005
BMP4 and Madh5 regulate the erythroid response to acute anemia
Blood Lenox et al.
105: 2741
Supplemental materials for: Lenox et al, Vol 105, Issue 7, 2741-2748
Files in this Data Supplement:
- Figure S1A. Staining f/f blood smears for siderocytes. (PDF, 39 KB) -
Staining of a blood smear from a newborn f/f mouse with Prussian Blue stain to identify iron granule in the fetal erythrocytes. Blue dots in the erythrocytes indicate the presence of siderocytes.
- Figure S1B. Genotyping the Sideroflexin mutation in f/f mice from our colony. (PDF, 65 KB) -
(Top) Schematic of PCR primers and the diagnostic cut sites used to genotype the Sfxn1 mutation in f/f mice from our colony. The bold sequence is region around the A insertion mutant reported by Fleming et al. Above and below this region are the sequences of the restriction sites used to differentiate between the alleles. Triangles mark the actual cut site. The asterisk above the C base represents an inserted C nucleotide that generates the diagnostic restriction sites. (Bottom) PCR analysis of DNA isolated from the mouse scored as a f/f in A. Both HinF1 and BsiE1 cut the PCR product, indicating that this animal is heterozygous for the A insertion in Sfxn1. The lower band indicated by the arrow is a non-specific amplification.
- Figure S1C. Direct sequencing of the Sfxn1 exon 2 in f/f mice. (PDF, 10 KB) -
Sfxn1 exon 2 from the mouse in A was PCR amplified and cloned into pTopo. Multiple independent plasmids were sequenced. Two sequences were obtained as marked in the boxes, mutant – GAATC and wild type – GATC.
- Figure S1D. Analysis of Sfxn1 mutations in f/f mice by Oligonucleotide ligation assay. (PDF, 80 KB) -
Oligonucleotide ligation assay (OLA) was used to confirm all PCR genotypes. (i) The technique utilizes a common anchoring oligo that is phosphorylated at its 5' end and biotinylated at its 3' end. Allele specific oligos that contain either a digoxigenin modification (wild type) or a fluorescein (mutant) at its 5' end. (ii) Ligation of the anchoring oligo to the allele specific oligo can only occur when the allele specific oligo binds the correct sequence. (iii) Anti-Dig conjugated to alkaline phosphatase, and anti-Fitc conjugated to HRP are used to detect the ligated products by ELISA. We tested all f/f mice in our colony by the PCR assay and OLA. Both assays gave identical results. We conclude that in our colony, the mutation in Sfxn1 reported by Fleming et al has been separated by recombination from the f/f mutant phenotype. We have identified numerous mice that exhibit the f/f phenotype, but are heterozygous for the Sfxn1 mutation. The f locus therefore cannot encode Sfxn1.OLA was done as described in Single-well genotyping of diallelic sequence variations by a two-color ELISA-based oligonucleotide ligation assay. Tobe VO, Taylor SA, and Nickerson DA. Nucleic Acids research 24:3728-2732, 1996.
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