Biology Reference
In-Depth Information
Transgene Methylation Analysis by Bisulfite Sequencing
Bisulfite treatment of DNA followed by sequencing was used to determine the en-
dogenous transgene promoter pattern of methylation in CTX0E03 cells. Methy-
lation is implicated in repression of genetic activity and involves the addition of
a methyl group to the carbon-5 position of cytosine residues of the dinucleotide
CpG. Bisulfite sequencing is a technique used to identify specific gDNA methyla-
tion sites based on the fact that treatment of DNA with bisulfite converts cytosine
residues to thymidine, but leaves 5-methylcytosine residues unaffected. Bisulfite
treatment thus introduces a specific change in the DNA sequence which is depen-
dent on the methylation status of individual cytosine residues. A bisulfite reaction
was performed using Epitect Bisulfite (Qiagen). Up to 2
µ
g of genomic DNA was
used for conversion with the bisulfite reagent. Bisulfite-converted DNA was used
as a template for PCR amplification. PCR primers were designed using the web
program METHPRIMER ([31]; http://www.urogene.org/methprimer/index1.
html webcite). Primers used were: Forward - GGGAGTTTGTTTTGGTAT-
TAAAATTAA and Reverse - ACTACTACTAATAAAAATTCTCCTCCTC.
PCR conditions were 94°C for 5 minutes, 30 cycles of 94°C for 30 seconds, 60°C
for 1 min, and 72°C for 30 seconds followed by 10 min at 72°C. PCR fragments
were cloned into TA-cloning vector using TOPO TA Cloning Kit for Sequencing
(Invitrogen). Ten individual clones were sequenced by GATC Biotech (Germany)
using the T3 universal PCR primers. The resulting sequences were analyzed us-
ing blast software http://blast.ncbi.nlm.nih.gov/Blast.cgi. Sequence analysis that
identified an unchanged cytosine base (i.e. protected from the bisulfite conversion
reaction) was considered to be methylated, but a cytosine conversion to thymi-
dine in the sequence indicated no methylation.
In Vitro Characterisation: Western Blot and
immunocytochemical Analysis
In vitro, CTX0E03 cell samples were prepared by maintaining the cells in non-
growth permitting medium alone, without the addition of EGF, bFGF, and
4-OHT, for 1- and 4-weeks. Western blot analysis for c-mycER
TAM
protein in
CTX0E03 cells was carried out using the whole-cell lysate. To prepare cell lysate,
CTX0E03 cell monolayers in a T75 flask were rinsed with cold PBS (4°C), lysed
with 750
µ
l 1× SDS Sample Buffer and dithiothreitol (DTT) Reducing Agent
(PAGEgel Inc, AMS Biotechnology, UK), and collected in a 1.5 ml centrifuge
tube. Samples were triturated multiple times to lyse and shear the sample, heated
at 100°C for 2 min and centrifuged. Cell lysates were loaded onto a 4%-20%
Tris-Tricine gel (PAGEgel Inc, AMS Biotechnology, UK), 10
µ
l per well. After
electrophoresis, the protein was transferred onto a nitrocellulose membrane with
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