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Supplemental Materials and Methods
Preparation of metallothionine-1 (MT-1) transgene construct
To produce transgenic mice expressing MT and luciferase specifically in the prostate, we
generated a 3.5-kb MT construct for microinjection (below). The construct consists of modified
prostate-specific rat probasin promoter ARR2PB (Zhang et al., 2000), which is linked to QBI
SP163 translational enhancer (Stein et al., 1998), followed by myc epitope-tagged mouse MT1
coding sequence (Reference Sequence: NM_013602.3). The elements were further downstream
coupled with internal ribosome entry site (IRES) for alternative translation initiation via this site
in the transcript and downstream linked to the luciferase gene with polyA signal from pGL3b
vector (Promega, Madison, WI). The sequence of this construct is shown in Table S1.
Specifically, ARR2PB promoter was HindIII/BamHI double digestion-released from ARR2BPmMT1/pGEM-3Z plasmid, a gift from Dr. Susan Kasper at the University of Cincinnati; the
SP163 element was PCR amplified with primer SP163F/SP163R (Table S2) using
pcDNA4/HisMax/LacZ plasmid as the template (Invitrogen, Carlsbad, CA); mouse MT1-myc
tag was generated by PCR with primer MT1F/MT1R using ARR2BP-mMT1/pGEM-3Z as the
template; IRES was KpnI/BglII double digestion-released from IRES/pGL3b plasmid (Zhang et
al., 2010). The above elements were sequentially ligated and cloned to pGL3b vector. In some
sequential enzyme digestions, calf intestinal alkaline phosphatase (CIP) was used for endspecific ligation, and gel electrophoresis was used to isolate specific fragments from plasmid and
PCR, Finally, the blunt-end 3.5kb MT1 construct was released from the plasmid by
EcolCRI/PshAI double digestion and gel purification.
Characterization of MT1 transgene construct in LNCaP cells
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MT1 construct and pGL3b plasmid control, together with the transfection internal control CMVLacZ plasmid were transfected to human prostate cancer cell LNCaP (ATCC, Manassas, VA), a
human prostate cancer cell line with androgen-receptor expression, as previously described
(Zhang et al., 2007). Luciferase and β-galactosidase activity assays were performed as reported
(Zhang et al., 2007). R1881, a synthetic androgen (Sigma, St. Louis, MO) was used to treat
transfected cells. Total RNA was extracted from cells in six-well plates by Trizol reagent
(Invitrogen). Reverse transcription was performed with the SuperScript III kit (Invitrogen) with 1
µg of total RNA in a 20 µl reaction mixture. For RT-PCR, 1 µl of cDNA was used in a 25 µl
PCR reaction mixture with the annealing temperature at 58 C in 35 cycles. Primers targeting
MT1-myc and GAPDH are shown in Table S2.
Production of transgenic (TG) mice
The heterozygous mice were produced by microinjection of the 3.5 kb MT construct into the
pronucleus of fertilized eggs from C57BL/6  SJL F2 hybrid mouse in the University of
Massachusetts Transgenic Animal Core Facility. The production of founder mice was
characterized by genotyping (below). Heterozygous transgenic mice (TG/WT) were maintained
by breeding with wild type (WT) C57BL/6J (Jackson Laboratory, Bar Harbor, ME). From
independent mouse founders, homozygous transgenic mice were produced by the heterozygous
transgenic mouse  heterozygous breeding scheme. Genotyping-positive transgenic offspring
(F1, homozygous [TG/TG], or heterozygous) were crossed with wild-type mice followed by
genotyping of at least 12 F2 pups to identify if the transgenic F1 mice were the homozygous
genotype. If the genotyping of all the pups was positive, the transgenic mouse was considered
homozygous. A homozygous mouse colony was maintained by breeding among homozygous
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mice. All transgenic mice were verified by genotyping.
Genotyping
A tail tip was cut from a newborn mouse for genotyping to identify transgenic or wild-type mice.
A 40 µl volume of extraction buffer (0.2% SDS, 0.1 M NaCl, 50 mM Tris, 25 mM EDTA, pH
8.0) and 10 µl 20 mg/ml of proteinase K (Invitrogen) was added to a tail segment for 54 C
overnight in O-ring sealed tubes. The digested tail was heated at 95 C for 5 min and centrifuged
for 1 min. One µl supernatant was taken as the template for 25 µl PCR reaction mixture in the
presence of DMSO and Platinum Taq (Invitrogen) according to the provider’s recommendation
for 37 cycles, with the annealing temperature at 59 C. TG mouse-specific primers
SP163F/mycR were used for the genotyping. In addition, primers targeting the luciferase gene
were also used to confirm the integrity of the inserted MT construct. The primer information is
listed in Table S2.
Luciferase expression in transgenic mouse tissues
A Precellys 24 homogenizer (Bertin Technologies, France) was used to homogenize mouse
tissue (5–20 mg, 11.5 weeks) in the presence of 0.5 ml 1 cell culture lysis reagent from the
luciferase assay kit (Promega). All tissues except bladder were homogenized for 15 sec, with
bladder homogenized for 30 sec, at 5000 rpm. To analyze luciferase activity, the homogenized
tissues were centrifuged for 1 min with 10 µl of supernatant taken for the luciferase assay as
previously described (Zhang et al., 2007). The expression of luciferase was normalized for the
weight of each tissue.
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Real-time qRT-PCR
Total RNA was isolated from ventral prostates using the mirVana miRNA Isolation kit (Life
Technologies, Carlsbad, CA) and reversed transcribed into cDNA using Supercript III (Life
Technologies) according to manufacturer instructions. The qPCR was performed using SYBR
GreenER qPCR SuperMix Universal (Life Technologies) as previously described (Tam et al,
2007). Melting curve analysis was performed to confirm the specificity of PCR reactions.
Relative expression of each gene was analyzed by CT method. Data were normalized to a
housekeeping gene, beta-2-microglobulin. The expression levels of untreated control ventral
prostates of 12.1MT1 mice were arbitrarily presented as 100 (as a reference value). Primer
information of transgene MT1, endogenous MT1 and beta-2-microglobulin is shown in
Supplemental Table S2.
References
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endothelial growth factor mRNA by internal ribosome entry: implications for translation under
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2. Tam N.N., Leav I., Ho S.M., 2007. Sex hormones induce direct epithelial and inflammationmediated oxidative/nitrosative stress that favors prostatic carcinogenesis in the Noble rat. Am J
Pathol 171: 1334-1341.
3. Zhang,J., Thomas,T.Z., Kasper,S., Matusik,R.J., 2000. A small composite probasin promoter
confers high levels of prostate-specific gene expression through regulation by androgens and
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glucocorticoids in vitro and in vivo. Endocrinology 141, 4698-4710.
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