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Blood, Vol. 107, Issue 1, 63-72, January 1, 2006
Long-term immune reconstitution in RAG-1-deficient mice treated by retroviral gene therapy: a balance between efficiency and toxicity
Blood Lagresle-Peyrou et al.
107: 63
Supplemental materials for: Lagresle-Peyrou et al
Files in this Data Supplement:
- Figure S1 (PDF, 37.1 KB) -
A. Schematic representation of retroviral vector. The location of the probes and primers has been noticed. B. Southern blot analysis of genomic DNA from various organs. Mouse A had developped the lymphoproliferative syndrome. Mouse B presented a total immune reconstitution. BM= bone-marrow. 10 µg of genomic DNA was digested with KpnI enzyme and Southern blot was performed according to standard protocols. DNA was hybridized with a [
 -32P dCTP] probe located in the cDNA of human RAG-1. The resulting hybridizing bands were visualized using a Molecular Dynamic Storm 860 Phosphoimager and its accompanying software. C. Northern blot analysis on the packaging cells. RNA was isolated from the non transfected and hRAG-1 transfected packaging cells and 5 µg was electrophoresed in formaldehyde agarose gel. The membrane was hybridized with the same probe as used for the Southern blot analysis. The resulting hybridizing bands were visualized and quantified using a Molecular Dynamic Storm 860 Phosphoimager and its accompanying software. RNA loading was evaluated after Ethidium bromide staining of the gel.
- Figure S2. Murine RAG-2 (mRAG-2) expression was determinated on the RNA extracted from thymus, spleen or bone marrow of a wild-type C57BL/10 mouse and the leukemic mouse (PDF, 11.2 KB) -
mRAG-2 expression was measured following the protocol described in materials and methods for the quantification of hRAG-1. To account for variations due to RNA extraction and the RT reaction, the measured level of mRAG-2 transgene mRNA was normalized against an endogenous RNA control, PO mRNA, an acidic ribosomal phosphoprotein ubiquitly expressed in all murine cells. Ratios were calculated by comparing the mRAG-2 mRNA expression levels (mRAG-2/PO) in the tested samples with murine RAG-2 expression levels in the thymus of wild type C57BL/10 mice (mRAG-2/PO). The mRAG-2 transcript expression value was calculated as a percentage of the reference mRAG-1/PO ratio in the thymus.
% mRAG-2 expression = (mRAG-2/PO) in the tested sample/(mRAG-2/PO) in WT mouse × 100
ND: not done in the organ specified.
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