Acute myeloid leukemia is associated with retroviral gene transfer to hematopoietic progenitor cells in a rhesus macaque
Blood Seggewiss et al.
107: 3865
Supplemental materials for: Seggewiss et al
Supplemental Materials and Methods
Histological Analysis. Immunhistochemistry was performed on formalin-fixed and paraffin-embedded tissue using polyclonal 
MPO, kappa and lambda antibodies and monoclonal MIB-1, CD20, CD45, and CD68 (KP1) (DakoCytomation, Carpenteria, CA), and monoclonal CD3 (SP7) (LabVision, Fremont, CA). Envision Plus detection system (DakoCytomation) with DAB as chromogen was used on a DakoAutomated immunostainer. GFP staining was performed using a rabbit polyclonal antibody (Santa Cruz, CA).
Cloning of integration sites. LAM-PCR and cloning of insertion site vector genomic fusion sequences was performed as described1,2.
Integration site specific PCR. The PCR analysis was performed on 100ng DNA with the primers for Chr. 15 and Chr. 9 as described3.
Southern Blot. 10µg DNA was digested with Nhe1 which cuts once in each LTR. The copy number was estimated by comparison to Jurkat clones with defined copy number of pCL20cMpGFP applying a phosphoimager scanner. For the detection of proviral integrants into the blast cell infiltrates in the kidney, 10µg DNA was digested with EcoR1 which cuts once in the proviral genome. The Southern blot was performed with a p32 labeled 670bp eGFP probe following standard procedures.
PALM® Single Cell Laser Microdissection, and Analysis. Laser dissection was carried out on 8µm formaldehyde fixed kidney cuttings mounted on PALM® MembraneSlides (P.A.L.M., Germany) followed by either HE or MPO staining. Slides were covered by PAM® LiquidCoverGlass (P.A.L.M., Germany). Circumcised cells were catapulted into PCR tube lids (Biozym, Germany) containing ProteinaseK, PCR buffer (Qiagen, Germany) and Igepal (Sigma, Germany).
PCR on gag region of retroviral competent retrovirus. The PCR analysis was performed on 200ng DNA with primers as described4.
BCL2-A1 expression analysis. Whole RNA was extracted using chloroforme/trizole, transcribed into cDNA using Superscipt™ First-Strand Synthesis System (Invitrogen, CA) for RT-PCR. Primers for the TaqMan BCL2-A1 RT PCR were as follows: 5’-TCTCTGGTCCGTAGTGTTACTTG-3’ and 5’-CGGCATCATTAACTGGGGAAG-3’. The FAM labeled probe was TaqMan® Gene expression assay with celera gene ID hCG201186 from Applied Biosystems, CA. Loading was controlled by comparing BCL2-A1 expression to actin expression. Actin primers were 5’-GGATCAGCAAGCAGGAGTATGA-3’ and 5’-GCGCAAGTTAGGTTTTGTCAAG-3’, the FAM-labeled probe was TCCATCGTCCACCGCAAATGCT. All samples were run in duplicates, plasmid standards in triplicates on ABI Prism 7900HT.
References
1. Schmidt M, Zickler P, Hoffmann G, et al. Polyclonal long-term repopulating stem cell clones in a primate model. Blood. 2002;100:2737-2743.
2. Calmels B, Ferguson C, Laukkanen MO, et al. Recurrent retroviral vector integration at the Mds1/Evi1 locus in nonhuman primate hematopoietic cells. Blood. 2005;106:2530-2533.
3. Kelly PF, Donahue RE, Vandergriff JA, et al. Prolonged multilineage clonal hematopoiesis in a rhesus recipient of CD34 positive cells marked with a RD114 pseudotyped oncoretroviral vector. Blood Cells Mol Dis. 2003;30:132-143.
4. Kelly PF, Vandergriff J, Nathwani A, Nienhuis AW, Vanin EF. Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein. Blood. 2000;96:1206-1214.
-actin PCR.