Supplemental methods
Southern blot analysis
Southern blot analysis was performed as previously described (Justice et al
1994). To analyze rearrangements of the B- and T-cell receptor loci in Prdm14
induced tumors, probes to IgH, IgK, J1, and J2 were utilized. Probes of 1.3-kb
(IgH), 1.5-kb (IgK), 1.9-kb (J1), and 1.2-kb (J2) were generated by PCR
amplification using the following forward and reverse primers: (5'GGTTAAAAATAAAGACCTGGAGAGG-3'), (5'TTTAGTATAGGAACAGAGGCAGAACA-3') for IgH; (5'GGGTTTTTGTACAGCCAGACA-3'), (5'-CATGAAAACCTGTGTCTTACACATAA3') for IgK; (5'-CCTCTCTTCACCCCTTAAGATT-3'), (5'GGGAAGGGACGACTCTGTCT-3') for J1; and (5'GAGGAAGGTGACGAAAGAGGA-3'), (5'-ATTTGGGTGGAAGCGAGAG-3') for
J2. Unrearranged germline loci were detected by IgH, IgK, J1, and J2 probes
as a 6.2-kb EcoRI fragment, a 4-kb BglII fragment, a 5.8-kb PvuII fragment, and
a 4.8 HindIII fragment, respectively. When no rearrangements were observed in
tumors, additional digests with XbaI (J1, J2) and BglI (IgH) were performed to
verify that a rearranged band was not co-migrating with the germline band. To
detect Prdm14-MIGR1 or MIGR1-EV vector integrations, a GFP probe was
generated by digesting MIGR1 with NcoI and HindIII. This probe was hybridized
to KpnI-digested genomic DNA to detect the number of integration events.
Northern blot analysis
Total RNA was prepared from cells or snap frozen tissues with RNA-STAT60
using the manufacturer’s instructions (Tel-Test Inc., Friendswood, TX). Ten mg
of total RNA was electrophoresed on a 1% agarose gel using Northernmax 10X
gel denaturing solution (Ambion) in 1x MOPS. Gel was transferred to Hybond N+
membrane (Amersham Biosciences, Piscataway, NJ) using an alkaline transfer
buffer (3M NaCl, 0.01 M NaOH) followed by UV-crosslinking. The membrane
was hybridized as previously described using XhoI digestion of the Prdm14MIGR1 vector to obtain the Prdm14 probe. The same GFP probe as was used
for Southern analysis was used for northern analysis (Dettman and Justice
2008).
Complete Blood Cell Counts
Whole blood was obtained from the retro-orbital sinus and placed into K2EDTA
tubes (Becton, Dickinson and Company [BD], Franklin Lakes, NJ). Complete
blood cell counts (CBCs) were analyzed using a Cell Dyn 3700R Veterinary
Hematology Analyzer (Abbott, Abbott Park, IL).
RNA purification
From mice, RNA was harvested from frozen tissue and from sorted cells with
RNAqueous Micro (Ambion) according to the manufacturer’s instructions. RNA
concentration and purity were confirmed using Eppendorf Biophotometer Plus
(Hamburg, Germany). cDNA was produced using Multiscribe reverse
transcriptase (ABI) with random hexamers.
RNA was extracted from human cell lines in log phase using RNA STAT60
(Tel-Test Inc.), and RNA from human samples was extracted from Ultraspec, per
manufacturer’s instructions. Human samples underwent selection for
polyadenylated mRNA using the MicroPoly(A)Purist™ Kit (Ambion). Samples
were treated with DNAse I, amplification grade (Invitrogen). cDNA was produced
as above, except with oligo d(T) priming, and was then treated with RNAse H
(Invitrogen).
Human cell lines
Jurkat cells (ATCC, Manassas, VA) were grown in RPMI with 10% fetal bovine
serum (FBS) at 37C in 5% CO2 and served as a negative control for PRDM14
expression. PA-1 ovarian teratocarcinoma cells (ATCC) served as a positive
control for PRDM14 gene expression (Nishikawa et al 2007) and were grown in
DMEM plus 10% FBS under the same conditions.
Dettman EJ, Justice MJ (2008). The zinc finger SET domain gene Prdm14 is overexpressed in
lymphoblastic lymphomas with retroviral insertions at Evi32. PLoS ONE 3: e3823.
Justice MJ, Morse HC, 3rd, Jenkins NA, Copeland NG (1994). Identification of Evi-3, a novel
common site of retroviral integration in mouse AKXD B-cell lymphomas. J Virol 68: 1293-1300.
Nishikawa N, Toyota M, Suzuki H, Honma T, Fujikane T, Ohmura T et al (2007). Gene
amplification and overexpression of PRDM14 in breast cancers. Cancer Res 67: 9649-9657.