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Blood, Vol. 106, Issue 7, 2530-2533, October 1, 2005
Recurrent retroviral vector integration at the Mds1/Evi1 locus in nonhuman primate hematopoietic cells
Blood Calmels et al.
106: 2530
Supplemental materials for: Calmels et al, Vol 106, Issue 7, 2530-2533
Files in this Data Supplement:
- Table S1. Primer sequences for LAM-PCR and RIS-specific PCR and Taqman (PDF, 80.7 KB) -
RNA analysis methodology: Total RNA was extracted from CD34+ cells, individual CFUs-GM, or purified granulocytes or mononuclear cells with the PicoPure RNA Isolation Kit (Arcturus, Mountain View, CA). A first round of cDNA synthesis was performed (Invitrogen, Carlsbad, CA) with 400 ng of total RNA and 1 µL of 5-µL T7-Oligo(dT) promoter primer, then amplified into cRNA with the MEGAscript T7 Kit (Ambion, Austin, TX). cRNA (200-400 ng) was used for the second round of cDNA synthesis with random primers. Thirty-five–cycle PCR was performed on 1.5 µL of cDNA, using primers EVI1-ex5-F1 with EVI1-ex6-R1 to amplify both EVI1 and MDS1-EVI1 transcripts, and MDS1-ex2-F1 with EVI1-ex2-R1 to amplify MDS1-EVI1 transcripts only.
- Figure S1. LAM-PCR site integration analysis of peripheral blood clonal patterns (PDF, 636 KB) -
Early (4-6 months) and late (most recent follow-up, median 70 months) for animals with MDS1/EVI1 RIS. MPT indicates months after transplantation. The arrow indicates the internal vector 3[prime] LTR amplification product. Time-course analysis shows no progression towards oligo- or monoclonality despite the presence of MDS1 insertions in each animal.
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