SUPPLEMENTARY INFORMATION
Supplementary Figure 1. Tandem repeats in the TSTA elements are not deleted.
HEK293 (293), HT-29, Hep G2, NIT-1, and -TC-6 cells were transduced with the
indicated vectors. Genomic DNA was isolated and PCR was performed with primers
designed to amplify the 345 bp of the two copies of the VP16 immediate early
transactivator in Gal4VP2 (a) or the 170 bp fragment containing the five copies of the
Gal4 DNA binding sites in G5E4T (b).
Supplementary Figure 2. Detection of insulin mRNA in cell lines. RNA was isolated
from the indicated cell lines or from primary islets as positive control. RT-PCR was
performed using primers to detect murine insulin I or II mRNA in murine insulinoma cell
lines (top panel), human insulin mRNA in human cell lines, or rat insulin mRNA in ARIP
cells (bottom panel). No RT was used in some reactions containing primary islets as
negative controls.
Supplementary Figure 3. Diagram of names and locations of restriction enzymes
used in Southern blot of Figure 2. The restriction enzymes used to digest genomic
DNA for Southern blot analysis and their approximate locations are indicated on each
lentiviral vector. The expected band of DNA (Kb) resulting from the enzyme digestion is
shown to the right.
Supplementary Materials and Methods
Polymerase chain reaction. Genomic DNA was isolated using a standard
phenol/chloroform extraction procedure or a DNeasy Tissue Kit (Qiagen; Valencia, CA)
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from transduced cell lines carrying an integrated vector copy of 1 or less. To detect the
170 bp fragment encompassing the five Gal4 DNA binding sites, 1-2 L of genomic DNA
was used per 50 L PCR reaction containing 2.0 mM MgCl2 using a sense primer (5’GCTCGCGAAAGCTTGCAT-3’) and an antisense primer (5’GCCAAGTGCAGAGCGAGTAT-3’) with the following conditions: 1 cycle at 94° C for 15
min, followed by 25 cycles at 94° C for 1 min, 59.2° C for 45 s, 72° C for 1 min, and a
final extension at 72° C for 10 min. To detect the 345 bp fragment containing the two
copies of the VP16 domain, 1-2 L of genomic DNA was used per 50 L PCR reaction
containing 2.5 mM MgCl2 using a sense primer (5’-GTTGACTGTATCGCCGGAAT-3’)
and an antisense primer (5’-ACAAAGATCCCAAGCTGTCG-3’) with the following
conditions: 1 cycle at 94° C for 15 min, followed by 25 cycles at 94° C for 1 min, 60° C
for 45 s, 72° C for 1 min, and a final extension at 72° C for 10 min.
Reverse-transcription PCR. Total cellular RNA were isolated using an RNeasy Mini Kit
(Qiagen, Valencia, CA), treated with DNase (Sigma-Aldrich; St. Louis, MO), and 100-200
ng RNA was reverse transcribed using a SuperScript First-Strand Synthesis System for
RT-PCR Kit (Invitrogen; Carlsbad, CA) with Oligo(dT) according to manufacturer’s
instruction. Five microliters of the resulting reaction containing the cDNA was subjected
to PCR. To detect a 260 bp human insulin product, PCR was performed in 50 L
reaction volume containing a sense primer (5’-TGTGAACCAACACCTGTG-3’), an
antisense primer (5’-CGTCTAGTTGCAGTAGT-3’), and 5 L of 10X Combination Buffer
(Gene Choice; Frederick, MD) with the following conditions: 1 cycle at 94° C for 15 min,
followed by 25 cycles at 94° C for 1 min, 53° C for 1 min, 72° C for 2 min, and a final
extension at 72° C for 10 min. To detect a 187 bp rat insulin product, PCR was
performed in 50 L reaction volume containing a sense primer (5’-
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CCTGCCCAGGCTTTTGTCAA-3’), an antisense primer (5’CTCCAGTGCCAAGTCTGAA-3’), and 5 L of 10X Combination Buffer (Gene Choice,
Frederick, MD) with the following conditions: 1 cycle at 94° C for 10 min, followed by 25
cycles at 94° C for 1 min, 60° C for 45 s, 72° C for 1 min, and a final extension at 72° C
for 10 min. To detect murine insulin sequences, PCR was performed in 50 L reaction
volume containing 2.5 mM MgCl2, forward primer (5’-GCAAGCAGGTCATTGTTTCA-3’)
and reverse primer (5’-GCTGGTAGAGGGAGCAGATG-3’) for a 329 bp murine insulin 1
product and forward primer (5’-TTTGTCAAGCAGCACCTTTG-3’) and reverse primer (5’TCTACAATGCCACGCTTCTG-3’) for a 241 bp murine insulin 2 product with the
following conditions for both insulins: 1 cycle at 94° C for 15 min, followed by 35 cycles
at 94° C for 30 s, 63° C for 30 s, 72° C for 1 min, and a final extension at 72° C for 10
min..
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