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Blood, Vol. 111, Issue 3, 1138-1146, February 1, 2008
The role of Ikaros in human erythroid differentiation
Blood Dijon et al.
111: 1138
Supplemental Materials for: Dijon et al
Files in this Data Supplement:
- Table S1. Sequences of primers used in construction of vectors or in quantitative PCR analysis (DOC, 54 KB)
- Table S2. Phenotype of erythroid cells during differentiation (DOC, 76 KB)
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On indicated days, cells were harvested and analysed by FACS for different cell surface marker (erythroid markers : CD36, CD71, GPA and CD111 ; progenitor markers : CD117 (c-kit), CD34 ; human hematopoietic marker : CD45 ; myeloid markers : CD33 and CD13). Percentage and number of cells are shown with mean ± SEM for 5 to 7 independent experiments.
- Figure S1. Recombinant lentiviral vectors (DOC, 109 KB)
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The lentiviral genome is represented here. Transgene expression is driven by the ubiquitous EF1  promoter. The EGFP transgene of pTRIP  u3 EF1 EGFP vector 36, also called EF1 GFP, was replaced with the Ik6 cDNA containing a Flag sequence (F). This new recombinant lentiviral vector was denominated EF1 IK6. LTR: Long Terminal Repeat; TRIP: Central Termination Sequence (CTS) and central PolyPurine Tract (cPPT) of HIV-1 virus; U3: deletion of 400bp in the 3′LTR U3 region encompassing promoter/enhancer elements of native HIV-1 virus (Self Inactivated vector).
- Figure S2. Expression of transgenic Ik6 in transduced (DOC, 75 KB)
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CB cells. On days 10, 13, 17 and 20, transgenic Ikaros 6 expression relative to endogenous GAPDH expression was analyzed by quantitative PCR in transduced erythroid cells derived from CD34+ CB cells. Expression of transgenic Ik6 was compared to expression of endogenous Ikaros. Results are shown as mean ± SEM for 4 to 7 independent CB samples.
- Figure S3. Gene deregulation in EF1 IK6 erythroid cells (DOC, 430 KB)
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PCR were performed using cDNA from transduced (EF1 GFP and EF1 IK6) erythroid cells on day 17 and 20. We obtained Ct values which are a measure of the amount of template cDNA; the lower the Ct value, the higher the amount of cDNA template. Ct value obtained for each gene was normalized with four endogenous genes (GAPDH, HRP1, Ubiquitin and â-Actin). We compared ratios obtained from EF1 IK6 cells with ratios obtained from EF1 GFP cells. We analyzed 184 genes, but only gene ratios with similar variations in three experiments were used. Ratios higher than 2 indicate that the gene is up-regulated. Ratios lower than −2 indicate that the gene is down-regulated. Ratios ranging between −2 and 2 indicate that gene expression was unchanged. (A) Analysis of apoptosis genes. Ten genes were up-regulated, 13 genes were down-regulated and 31 genes were unchanged with Ik6 over-expression. (B) Analysis of transcription factor, cell cycle, apotosis implicated genes. Thirteen genes were up-regulated, 17 genes were down-regulated and 20 genes were unchanged as a consequence of Ik6 forced expression.
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