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Blood, Vol. 109, Issue 6, 2630-2633, March 15, 2007
Distinct roles of Mdm2 and Mdm4 in red cell production
Blood Maetens et al.
109: 2630
Supplemental materials for: Maetens et al, Vol 109, Issue 6, 2630-2633
Files in this Data Supplement:
- Table S1. Loss of mdm2 in erythroid cells is lethal (PDF, 10 KB)
- Table S2. Loss of mdm4 in erythroid cells (PDF, 9.98 KB)
- Figure S1. The defects associated with specific inactivation of mdm2 in the erythroid lineage are p53 dependent (PDF, 22.9 KB) -
(A) mdm2lox/lox; EpoRGFP-Cre/+; p53–/– mice are viable. This table shows the genotyping distribution at weaning age of 17 mice generated from mdm2lox/lox; p53–/– × mdm2lox/+; EpoRGFP-Cre/+; p53+/– intercrosses. (B) Schematic representation of part of the mdm2 wild-type, floxed, and loxP alleles. Exons are represented by black boxes, the loxP sites by black arrows, and the gray arrows represent the position of the 2 primers used for the PCR analysis shown in panel C. (C) PCR amplification of genomic DNA from E10.5 yolk sac (YS) or E14.5 fetal liver (FL) preparations of embryos with the indicated genotypes. PCR were performed so that only the 226-bp fragment, indicating the presence of the loxP allele, is amplified. Normalization of the samples was performed using a primer pair that specifically amplifies a fragment of 250 bp of a nonrelevant (wild-type) locus. PCR amplifications were interrupted in the linear range and DNA was loaded on agarose gels. This analysis confirms efficient Cre-mediated recombination at the mdm2 floxed locus. E14.5 mdm2lox/lox; EpoRGFP-Cre/+; p53–/– embryos were indistinguable from control littermates (not shown), despite efficient excision of exons 5 and 6 in their fetal livers. Note that a significant increase in the amount of PCR products is observed in the yolk sac from p53–/– compared with p53 wild-type embryos. This observation is consistent with the rescue of the blood island-erythroid progenitor cell number in the absence of functional p53. l indicates lox; and B, blank (no DNA). (D) Hematocrits of E14.5 mdm2lox/lox; EpoRGFP–Cre/+; p53–/– (n=2; open squares) versus control mdm2lox/l+ or lox/lox; EpoR+/+ or GFP-Cre/+; p53+/– or p53–/– (n=9; filled diamonds) embryos. Whereas all mdm2lox/lox; EpoRGFP-Cre/+ embryos were resorbed at that stage of development, mdm2lox/lox; EpoRGFP-Cre/+; p53–/– embryos exhibited normal hematocrits, indicating that absence of p53 rescues the erythroid defects associated with mdm2 inactivation in the erythroid lineage.
- Figure S2. Cre-mediated recombination occurs at the mdm2 and mdm4 loci with comparable efficiencies (PDF, 31.3 KB) -
(A) Schematic representation of part of the mdm2 (left panel) and mdm4 (right panel) wild-type, floxed and loxP alleles. Exons are represented by black boxes, the loxP sites by black arrows, and the small arrows represent the position of the 3 primers used for the PCR analyses shown in panels B and C. (B) PCR amplification of genomic DNA from E10.5 yolk sac preparations of embryos with the indicated genotypes. PCRs were performed using primers a and c and so that only the 400-bp fragment, indicating the presence of the loxP allele, is amplified. Normalization of the samples was performed using a primer pair that specifically amplifies a fragment of 250 bp of a nonrelevant (wild-type) locus. PCR amplifications were interrupted in the linear range and DNA was loaded on agarose gels. This analysis confirms efficient Cre-mediated recombination at both mdm2 and mdm4 floxed loci. Note that the number of cycles necessary to obtain a significant signal in preparations from the mdm2 mutants (34 cycles) was higher than in mdm4 mutants (29 cycles). This is likely to reflect the proportional difference in the number of erythroid progenitor cells in the yolk sac of both mutants. (C) PCR amplification of genomic DNA from yolk sac preparations of embryos with the indicated genotypes. PCR were performed using primers a, b, and c. This analysis further confirms efficient Cre-mediated recombination at both mdm2 and mdm4 floxed loci. Note that the ratio floxed versus loxP allele in preparations from the mdm2lox/lox; EpoRGFP-Cre/+ was higher (80:20) than in the mdm4lox/lox; EpoRGFP-Cre/+ (50:50) embryos. This is likely to reflect the proportional difference in the number of erythroid progenitor cells in the yolk sac of both mutants. In contrast, in the yolk sac of mdm2lox/lox; EpoRGFP-Cre/+; p53–/– rescued embryos, which contain a number of erythroid progenitor cells comparable with wild-type controls, this ratio (50:50) is similar to that observed in mdm4lox/lox; EpoRGFP-Cre/+ embryos.
- Figure S3. Mdm4 is dispensable for adult erythropoiesis (PDF, 23.4 KB) -
(A) Number of CFU-Es, BFU-Es, and CFC-m’s detected in methylcellulose cultures supplemented with Epo, Epo and IL-3, or a mixture of IL-3, IL-6, and SCF, respectively. Numbers are expressed per 5 × 104 nucleated bone marrow cells for the CFU-E count, and 2 × 105 and 1.5 × 104 cells for the BFU-E and CFC-m counts, respectively. Data from controls (black; mdm4lox/+; EpoR+/+) and mutants (gray; mdm4lox/lox; EpoRGFP-Cre/+) represent the mean ( SD) of 6 independent experiments. (B) Schematic representation of part of the mdm4 wild-type, floxed, and loxP alleles. Exons are represented by black boxes, the loxP sites by black arrows, and the arrows represent the position of the primers used for the PCR analyses shown in panels C and D. (C) PCR amplification of genomic DNA from adult bone marrow preparations of mice with the indicated genotypes. PCRs were performed so that only the 400-bp fragment, indicating the presence of the loxP allele, is amplified. Normalization of the samples was performed using a primer pair that specifically amplifies a fragment of 250 bp of a nonrelevant (wild-type) locus. PCR amplifications were interrupted in the linear range and DNA was loaded on agarose gels. (D) Examination by PCR analyses of the ratio between the floxed allele (675 bp) and loxP allele (400 bp) from yolk sac (YS) and adult bone marrow preparations of mice with the indicated genotypes. Bone marrow cells from three adult mice were pooled and Ter-119–positive cells purified by MACS sorting. PCR, using a mixture of primers a, b, and c, were performed both on total bone marrow (input) and Ter-119–enriched (Ter119+) cell populations. PCR amplifications were interrupted in the linear range and DNA was loaded on agarose gels. The data show efficient Cre-mediated recombination at the mdm4 locus within the erythroid lineage of adult mdm4lox/lox; EpoRGFP-Cre/+ mice.
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