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Blood, Vol. 111, Issue 8, 4092-4095, April 15, 2008
Survivin overexpression alone does not alter megakaryocyte ploidy nor interfere with erythroid/megakaryocytic lineage development in transgenic mice
Blood McCrann et al.
111: 4092
Supplemental materials for: McCrann et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 72.9 KB)
- Figure S1. Establishment of two separate transgenic mice models that overexpress survivin in the MK/erythroid lineage (JPG, 91.6 KB)
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(A) Constructs for conditional survivin expression in the megakaryocyte lineage using the Tet-Off system. Previously described PF4-tTA transactivator mouse line expressing the transactivator protein tTA-VP16 under the platelet factor 4 promotor (PF4).2 Responder mouse (TRE-surv) expressing murine survivin cDNA and  -galactosidase under a bi-directional tet-responsive element using a minimal CMV promotor (TRE-bidirec-mCMV). (B) Structure of the GATA-1-surv transgene. The human survivin cDNA was inserted between 7 kb of GATA-1 upstream regulatory sequences and 1.5 kb of sequences downstream of exon 3. (C) Southern blot of PF4-tTA responder line showing detection of the transgene at the expected band size of 1.6 kb following digestion of tail DNA with BamH1. (D) Southern blot of TRE-survivin line 12 confirming the transgene with an expected band of 2.2 kb following PstI digest of tail DNA. (E) PCR-genotyping of progeny from a heterozygous TRE-survivin TG/+ mouse crossed with a PF4-tet TG/+ mouse. Survivin transgene specific primers produce a 300 bp PCR product and are unable to detect the endogenous gene. PF4-tet primers detect a 450 bp PCR product using primers to the tTA-VP16 transgene. The * indicates double transgenic progeny which are positive for both transgenes. (F) PCR confirmation of GATA-1-surv transgenes in 7 founder lines. No DNA control and vector control reactions are shown in lanes 2 and 3.
- Figure S2. Survivin overexpression in MK and Erythroid lineages does not influence cell populations of MKs, GR1/Mac myeloid cells, erythrocytes, and c-kit positive progenitors (JPG, 228 KB)
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(A) Flow cytometry analysis of MK, GR1/Mac, erythroid, and c-kit+ cells in 1 year-old GATA-1-surv and wild type BM did not reveal significant differences in lineage populations in these animals. (B) Cell suspensions isolated from spleen show similar results as in (A). Representative plots are shown.
- Figure S3. CFU-MK colony assays with GATA-1-surv BM display a decrease in MK number and MK colony size in comparison to wild type BM (JPG, 55 KB)
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Bone marrow from GATA-1-surv transgenic and Wt mice was isolated from femurs. Red blood cells were lysed, the remaining BM cells were plated in MegaCult-C for CFU-MK (30,000 per ml) and cultured for 7 days. Following incubation, MK colonies were identified and quantitated based on positive acetylcholinesterase activity. Large colonies were defined as those with greater than 50 cells, while small colonies were defined as those with fewer than 50 cells. The average numbers of colonies ± standard deviation are shown.
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