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Supplementary information
RNA isolation and reverse transcriptase polymerase chain reaction (RT-PCR)
Total RNA was extracted from 5 × 106 BMMNCs using Trizol (Invitrogen; Carlsbad, CA, USA) according
to the protocol supplied with the reagent. The cDNA was synthesized from 2 μg of total RNA by extension
with oligo (dT) primer in 20 μl reaction mixture containing 10 U of reverse transcriptase (GIBCO-BRL,
Karlsruhe, Germany). RT-PCR was performed using Premix Taq (Takara, Shiga, Japan). AML1a and
AML1b/1c transcripts were amplified by using primers as described previously 1. In order to identify linear
range of the amplification, reaction cycle in exponential growth of each gene was optimized 2. The
amplification cycles of AML1a and β-actin were 33 and 22 cycles, respectively. A leukemia cell line U937
was used as a positive control. As an internal control, a 271-bp fragment of the β-actin gene was also
amplified (22 cycles of 30s at 94 °C, 30s at 58 °C, and 30s at 72 °C). Densitometric analysis was carried
out using AlphaEaseFC 4.0 image analysis software. Expression of a target gene (R) was calculated against
β-actin in the same sample. To detect AML1a expression in mice, RT-PCR was performed with RNA of
4×106 spleen cells of the mice. The primers were: 5’-CACTATAGGGAGACCCAAG-3’ and 5’CCCTATTCTATAGTGTCACCT-3’. The amplification reaction was carried as following: 94°C for 5 min,
33 cycles of 94°C for 45s, 54°C for 45s and 72°C for 45s, and 72°C for 2 min. As an internal control, PCR
was also performed in parallel using primers for mouse β-actin.
Construction of plasmids
The pBlue-script II KS (+) plasmid containing AML1a cDNA was generously provided by Prof. Misao
Ohki. The AML1a cDNA without the start codon was amplified from the above plasmid by primer:
5’-CGGAATTCCGTATCCCCGTAGATGCC-3’ and 5’-CGGAATTCGCGGCCGCCTCTATCCTGGCTGG
GGAG-3’, and cloned into the EcoRI-site of pcDNA3-FLAG. The AML1a plus FLAG tag cDNA was
obtained by PCR from pcDNA3-FLAG-AML1a, and subcloned into blunt XhoI ends of the
pMSCV-IRES-YFP
vector.
The
following
primers
were
used:
5’-CTCGAGCACTATAGGGAGACCCAAG-3’ and 5’-CTCGAGCCCTATTCTATAGTGTCACCT-3’. The
fusion expression product is 258 amino acids of AML1a and FLAG polypeptide. The pCMV5-AML1b
plasmid was kindly provided by Dr. Hiebert. The expression product is 479 amino acids in length. The
sequence of -62 to -71 of the two report plasmids, pM-CSFR-luc luciferase promoter with wild
AML1-binding site and pM-CSFR (mB)-luc luciferase promoter with mutant AML1-binding site, is
TGTGGTTGCCT and TCTAAGGTACC, respectively. The reference plasmid pCMV-β-galactosidase was
purchased from Qiagen (Germany). All PCR products were directly ligated into the pMD18-T simple
vector (Takara) and sequenced.
Viral production, transduction and transplantation of murine bone marrow and tumor cell
transplantation
293T
cells
were
transfected
with
pMSCV-IRES-YFP,
pMSCV-FLAG-AML1a-IRES-YFP,
the
envelope-encoding plasmid pEco, or the packaging plasmid pGP using a method of calcium phosphate
precipitation. Culture medium was collected at 48 and 72 h after the transfection, and filtered with 0.45 μm
filters. Retrovirus titers were determined by transducing NIH3T3 cells (1×105) with serial dilutions of the
retrovirus in the presence of 8 μg/ml polybrene (Sigma, Deisenhofen, Germany) at 72 h after infection. The
titer was calculated by multiplication of the total number of YFP-positive cells with the dilution factor of
the retroviral supernatant. At 48 h post transduction, percentage of infected cells was determined by flow
cytometric analysis of YFP expression. Bone marrow cells were harvested from male C57BL/6J donor
mice 3 days after injection of 150 mg/kg 5-fluorouracil (5-Fu) (Sigma), and pre-stimulated overnight in
Iscove modified Dulbecco medium (IMDM)/20% FCS supplemented with 10 ng/ml murine IL-3 (mIL-3),
10 ng/ml mIL-6 and 50 ng/ml mSCF. All cytokines were purchased from R&D system. Cells were
transduced by 3 rounds of spin infection (1,200×g, 25°C, 90 minutes) every 24 hours in retroviral
supernatant supplemented with growth factors and 8 μg/ml polybrene. Cells were re-suspended in Hanks
balanced salt solution and injected into the tail vein of lethally irradiated (9Gy) female C57BL/6J mice.
Rate of YFP-positive cells in peripheral blood (PB) was closely monitored by FACS. 5×106 spleen cell
suspension of each leukemic mouse was administered to the recipient mice after an irradiation dose of 4.5
Gy. All animals were maintained in a special caging system with autoclaved food and water.
Supplementary Reference List
1.
Miyoshi H, Ohira M, Shimizu K, Mitani K, Hirai H, Imai T, et al. Alternative splicing and genomic structure of the
AML1 gene involved in acute myeloid leukemia. Nucleic Acids Res 1995; 23: 2762–2769.
2.
Becker S, Quay J, Soukup J. Cytokine (tumor necrosis factor, IL-6, and IL-8) production by respiratory syncytial
virus-infected human alveolar macrophages. J Immunol 1991; 147:4307–4312.