Supplementary Materials and methods

RT-PCR

One mg of the total RNA was subjected to reverse transcription using oligo-dT and PrimeScriptTM 1st strand cDNA kit (Takara, Shiga, Japan). PCR was performed using PrimeStar DNA polymerase (Takara, Shiga, Japan) and 5’-ACGGCCGTGTATTACTGTGC-3’ and 5’-GGCCACGCTGCTCGTATC-3’ primers set at VH and Cm, respectively. Two PCR products were cloned into pUC/118, and then nucleotide alignments were determined for ten clones.

Vector construction

Mouse genomic sequence including pre-miR-125b-1 or pre-miR-30a was PCR-amplified from C57BL/6 mouse genomic DNA with the following primers: 5’-TTTTGTGAAGGGGAGAGGTG-3’ and 5’-TGGAAGCCTCAAGGGTGTAT-3’ for pre-miR-125b-1 (422 bp), 5’-TCTAGGGCATCGAGGCTTTG-3’ and 5’-TTTTCCACCCAAGCTTTGTAA-3’ for pre-miR-30a (288 bp), respectively. The fragment was cloned into the EcoRI site of pEm/IGH plasmid, pMXsEF1-(GFP), or pMXsEF1-(puror). Expression fragments of IGH/miR-125b-1 were PCR-amplified from the cDNA of a tumor derived from BCP-ALL patient7 with the following primers: 5’- TGTGCAAGAACATGAAACACCT -3’ and 5’- AATTCTCACAGGGGACGAGG -3’. The 3’UTR of the mouse Trp53inp1 was PCR-amplified from C57BL/6 mouse genomic DNA with the following primers: 5’-GAATGTTGAGTTATGTTGGTTTG-3’ and 5’-ACTGACAGATTTAAAAACTCTGTGC-3’.

Real-time PCR

Mature miR-125a expression was measured using a miScript SYBR Green PCR kit (QIAGEN, Hilden, Germany). cDNA was amplified with miR-125a-specific primers (QIAGEN, Hilden, Germany). Expression of U6 small nuclear RNA, as an internal control, was used for normalization of the results. Human Blood, Peripheral Leukocytes Total RNA (Clontech, Palo Alto, CA, USA) was used as a normal control. For mRNA real-time PCR, the following primer pairs were used: 5’-CCACAGAGCAGTCAGTATTTGGA-3’ and 5’-CTGTTTCTCTCCCTTTCTTGTGG-3’ for Trp53inp1, 5’-AGAGGGAAATCGTGCGTGAC-3’ and 5’-CAATAGTGATGACCTGGCCGT-3’ for b-actin.

Southern blot analysis of the integration site of Em/miR-125b-TG mice

Seven mg of genomic DNAs were digested using BamHI or HindIII and subjected to Southern blot analysis using a transgene-specific, 488 bp DNA fragment obtained by PCR using 5’-GTTGTTTAGAATGGGAAGATGTCC-3’ and 5’-GTAGGAAAGAGAAGAAGGCATGAAC-3’.

Cloning of the integration site of Em/miR-125b-TG mice.

The integration site of the TG116 was cloned by nested inverse PCR using 5’-TGTTCATGCCTTCTTCTTTTTCCTACA-3’ and 5’-AAGAGGAGACAATGGTTGTCAACAGAG-3’ for the first PCR and 5’-TGGTTATTGTGCTGTCTCATCATTTTG-3’ and 5’-CCCATTCTAAACAACACCCTGAAAACT-3’ for the second PCR on circular DNA after Hind III digestion. The integration site of the TG005 was also cloned as in TG116 using circular DNA after BamH I digestion and 5’-TGAGACCCTAACTTGTGATGTTTACCG-3’ and 5’-AAGAGGAGACAATGGTTGTCAACAGAG-3’ for the first PCR and 5’-TAATACACCCTTGAGGCTTCCAGAATTC-3’ and 5’-CCCATTCTAAACAACACCCTGAAAACT-3’ for the second PCR.

Retroviral infection and stable cell lines

Retroviruses were generated by transient transfection of Plat-E packaging cells with Fugene 6 (Roche Diagnostics), as described.22 32Dcl3 cells were infected with retroviruses in the presence of 10 mg/ml Polybrene. Transduced cells were selected with puromycin at 1 mg/ml 48 h after infection.

Cell culture for apoptosis assay

Stable transfectants of 32Dcl3 cells with miR-125b expression vector (pMXsEF1-miR-125b-(puror)), miR-30a expression vector (pMXsEF1-miR-30a-(puror)), or mock (pMXsEF1-(puror)) were cultured in the culture medium (RPMI-1640 containing 10% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin) lacking IL-3.

Supplementary Figure legends

Supplementary Figure 1. Nucleotide alignment of the IGH/miR-125b-1 chimera sequence. The sequence was analyzed using web sites at The VDJsolver 1.0 server (http://www.cbs.dtu.dk/services/VDJsolver/) and the BLAST search program at (http://www.ncbi.nlm.nih.gov/BLAST/). The VH (IGHV4-61*03), DH (IGHD2-21*02), JH (IGHJ4*02) and Cm sequences are highlighted in yellow, blue, green, and red, respectively. The 11q24-derived sequence containing pre-miR-125b-1 is highlighted in gray. The underlined and small letters are primers and N-nucleotides, respectively. The italic and bold letters indicate the mature miR-125b sequence (TCCCTGAGACCCTAACTTGTGA).

Supplementary Figure 2. Analysis of the expression of miR-125a in patients with hematological diseases. Relative expression levels of miR-125a were examined by real-time PCR in PB cells derived from patients with hematological diseases. Data represent the means ± SEM of two independent experiments.

Supplementary Figure 3. Southern blot analysis and molecular cloning of the integration site of Em/miR-125b-TG mice. (A) A schematic of the transgene is shown. The transgene consists of a human intronic immunoglobulin heavy chain enhancer (Em), a mouse VH promoter, and a rabbit b-globin gene containing the truncated exon2 and the exon3. A 422 bp mouse genomic sequence containing pre-miR-125b-1 was inserted into the EcoRI site of the rabbit b-globin gene. (B) Southern blot analysis using transgene-specific DNA fragments as a probe showed different integration in TG116 and TG005. Solid and dotted arrows represent inserted transgenes in the tandem and chromosomal integration sites, respectively. (C) The integration site of the TG116 or TG005 was cloned by nested inverse PCR. The probe of Southern blot analysis used was a transgene-specific DNA fragment. Solid and dotted arrows represent inserted transgenes in the tandem and chromosomal integration sites, respectively. (D) Nucleotide alignments of integration points are shown. The transgene was integrated into chromosome 15 and 5 in TG116 and TG005, respectively. Uppercase letters represent the sequence of transgenes and lowercase letters represent the sequence of mouse chromosomes.

Supplementary Figure 4. Characteristics of morbid TG116 and TG005 mice. Spleen weight, liver weight, the number of BM cells, the number of white blood cells (WBCs), and concentrations of hemoglobin of morbid TG116 and TG005 mice developing B220+ tumors or WT mice are shown. P-values (*) of < 0.05 were considered significant using a 2-sample t test with Welch correction.

Supplementary Figure 5. Analysis of young TG116 mice. (A) BM cells derived from TG116 mice or WT mice four weeks after birth were analyzed for expression of CD19, IgM, CD43, CD3, and CD11b versus expression of B220 by FACS. (B) Southern blot analysis of BM cells derived from TG116 mice four weeks after birth (lanes 1-4). Clonal rearranged bands (arrows) were detected in lane 3. G, germ line control. P, positive control identical to the sample of lane 1 in Figure 2F.

Supplementary Figure 6. miR-125b inhibited apoptosis induced by IL-3 withdrawal in 32Dcl3 cells. (A) Relative expression levels of miR-125b were examined by real-time PCR in stable transfectants of 32Dcl3 cells with pMXsEF1-miR-125b-(puror) or mock (pMXsEF1-(puror)) (left side of the histograms). Relative expression levels of Trp53inp1 were examined by real-time PCR in these cells at different time intervals after IL-3 withdrawal (right side of the histograms). Data points correspond to the means ± SEM of three independent experiments. (B) Stable transfectants of 32Dcl3 cells transduced with pMXsEF1-miR-125b-(puror), pMXsEF1-miR-30a-(puror), or mock (pMXsEF1-(puror)) were cultured in the culture medium without IL-3. Cells were stained with FITC AnnexinV and Propidium Iodide (PI) at different time intervals after IL-3 withdrawal (left panel). Apoptotic cells, AnnexinV and PI positive cells, were measured by FACS (right panel). Data points correspond to the means ± SEM of three independent experiments. P-values (*) of < 0.05 were considered significant using a Student’s two-tailed t test. All data were representative of three independent experiments. N.D., not detected.

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