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Blood, Vol. 109, Issue 9, 3906-3914, May 1, 2007
Myeloproliferative disease induced by TEL-PDGFRB displays dynamic range sensitivity to Stat5 gene dosage
Blood Cain et al.
109: 3906
Supplemental materials for: Cain et al, Vol 109, Issue 9, 3906-3914
Files in this Data Supplement:
- Figure S1. TEL-PDGFRB retroviral constructs with add-back Stat5 protein expression (JPG, 38.2 KB)
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(A) Retroviral constructs encode TEL-PDGFRB followed by an internal ribosomal entry site to allow expression of a second cDNA, either Stat5a or Stat5b. (B) 293T cells were transiently transfected with a retroviral construct as indicated and probed for the protein as shown, using  -actin as a protein loading control. Detection of TEL/PDGFR protein using an antibody recognizing the C-terminus of native PDGFR. (C) Detection of Stat5 protein as shown, using antibodies that specifically recognize either Stat5a or Stat5b, as indicated. (ires indicates internal ribosomal entry site; GFP, green fluorescent protein; LTR, long terminal repeat; TPiGFP, TEL-PDGFRB ires eGFP; TPiStat5a, TEL-PDGFRB ires mStat5a; and TPiStat5b, TEL-PDGFRB ires Stat5b.)
- Figure S2. The number of retroviral integration sites is equivalent in TPiGFP→Stat5a−/− and TPiGFP→Stat5a+/+ recipient mice (JPG, 42.8 KB)
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(Lanes 1-3) TPiGFP→Stat5a−/− mice; (lane 4) TPiStat5a→Stat5a−/− mouse; and (lanes 5-6) TPiGFP→Stat5a+/+ mice. DNA was isolated from whole bone marrow cells of recipient mice and amplified by linear amplification-mediated (LAM)–PCR as described by Schmidt et al (Blood. 2002;100:2737-2743). Briefly, genomic DNA was amplified using 5′-biotinylated primer MLTR1 (5′-CTCAATAAAAGAGCCCACAACCC-3′). Amplified DNA was selected using magnetic beads conjugated to streptavidin, primed with a hexanucleotide mixture, and then fragmented using Tsp509I. A linker cassette was ligated to the fragmented DNA and then amplified using primer MLTR2 (5′-TCCTCCGATAGACTGCGTCG-3′) and linker cassette primer LC1 (5′-GACCCGGGAGATCTGAATTC-3′). DNA was denatured and amplified by nested PCR using primers MLTR3 (5′-GCCTCTTGCTGTTTGCATCC-3′) and LC2 (5′-GATCTGAATTCAGTGGCACAG-3′).
- Figure S3. Stat5a and Stat5b both restore myeloproliferation when coexpressed with TEL-PDGFRB in Stat5a deficient bone marrow cells (JPG, 49.3 KB)
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Kaplan-Meier graph depicts survival of mice that underwent transplantation, summarized in Table 1. Disease phenotype at time of death was characterized as either fatal MPD (WBC, > 50 × 109/L 50 000/µL and spleen weight, > 450 mg) or MPD (WBC, > 15 × 109/L 15 000/µL and/or spleen weight, > 450 mg), with number of analyzed mice indicated. TPiStat5a→Stat5a+/− mice developed severe MPD (spleen weight, 520 mg n = 1; WBC, 66 × 109/L 66 000/µL n = 1). Moderate MPD was detected in TPiStat5a→Stat5a−/− (spleen weight median, 800 ± 170 mg; range, 550-970 mg n = 8; WBC median, 82 ± 106 × 109/L 82 000 ± 106 000/µL; range, 40-108 × 109/L 40 000-108 000/µL n = 7) and TPiStat5b→Stat5a−/− (spleen weight median, 470 ± 184 mg; range, 340-600 mg n = 2; WBC median, 23 ± 10 × 109/L 23 000 ± 10 000/µL; range, 16-30 × 109/L 16 000-30 000/µL n = 2). (TPiGFP indicates TEL-PDGFRB ires eGFP; TPiStat5a, TEL-PDGRB ires mStat5a; TPiStat5b, TEL-PDGFRB ires Stat5b; MPD, myeloproliferative disease; and WBC, peripheral white blood cell count.)
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