Figure 8. Schematic of the methylated DNA immunoprecipitation (MeDIP) procedure. For a more detailed explanation, refer to
As an alternative, we developed and now use a methylated DNA immunoprecipitation (MeDIP) procedure.4 This procedure is illustrated in Figure 8: DNA is denatured and digested, and is incubated with an antibody that specifically
recognizes 5-methylcytidine. The resultant immune complexes are incubated with protein A/G beads, and following washing and
centrifugation steps, the captured material is eluted and can be subjected to endpoint PCR, or, if quantitation is desired,
to qPCR. The advantages of this procedure compared to a methylation-sensitive Southern blot are that it is high throughput,
requires no radioactivity, allows for quantitative results using qPCR, and additionally, through the use of specific PCR primers,
can enable a determination of which region(s) of DNA are methylated.
Figure 9. MeDIP assay results of 16-6F and subclone 16-6F A5. Genomic DNA from the 211 generation time point of the unstable
cell line 16-6F described by Figure 7 and associated text was subjected to the MeDIP procedure illustrated in Figure 8. Forward
and reverse PCR primers were specific for 5' and 3' sequences just upstream and downstream of the promoter region of the transfected
vector construct. The lane labeled "+ control" is in vitro methylated plasmid also subjected to the MeDIP procedure. 16-6F
was later re-cloned, and a resultant stable subclone, A5, was also subjected to the MeDIP procedure. The arrow marks the migration
of the methylated PCR product.
Figure 9 shows the results of a MeDIP experiment on the same 16-6F genomic DNA from the Southern blot experiment shown in
Figure 7. Endpoint PCR made use of primers directed to the promoter region of our transfected vector construct, and as expected,
the results showed no detectable PCR product from untransfected CHO cells. Also as expected, the lane labeled "+control" showed
a very strong signal for in vitro methylated DNA plasmid. The lane labeled "16-6F," which contained the MeDIP PCR product from the genomic DNA sample from the
phenotypically unstable 16-6F cell line prepared at 211 generations (recall from Figure 7 that this DNA was not fully digested
with the methylation sensitive enzyme AatII), showed a strong signal as well. Although the size of DNA fragments generated
during the digestion step of the MeDIP procedure needs to be considered, these latter results suggested that at least a portion
of the methylation is in the vicinity of the promoter region of our integrated transfected vector construct. The 16-6F cell
line was later re-cloned, and genomic DNA from a resultant high-expressing subclone, 16-6F A5, whose protein expression profile
was stable, was subjected to the same MeDIP and PCR assay conditions. Figure 9 shows that in the lane labeled "16-6F A5,"
there is a much reduced methylation signal. Taken together, these data indicated that recloning resulted in the isolation
of a clone having reduced levels of expression vector methylation, and that the markedly reduced methylation in this clone
correlated with its increased protein expression and increased stability.
The MeDIP assay allows us to increase our understanding of the methylation status of our integrated expression constructs.
By applying it broadly to our Phase 1 antibody projects, the MeDIP assay will enable us to monitor the extent to which epigenetic
changes of this nature occur in our cell lines and negatively affect the outcome of our cell line development efforts.
Robin A. Heller-Harrison is the associate director and the corresponding author in the cell and molecular sciences group in the department of Drug Substance Development at Wyeth Biopharma
Articles by Robin A. Heller-Harrison
Kerstin Crowe is a research scientist II in the cell and molecular sciences group in the department of Drug Substance Development at Wyeth Biopharma
Articles by Kerstin Crowe
Cecilia Cooley is a research scientist I in the cell and molecular sciences group in the department of Drug Substance Development at Wyeth Biopharma
Articles by Cecilia Cooley
Megan Hone is scientist II in the cell and molecular sciences group in the department of Drug Substance Development at Wyeth Biopharma
Articles by Megan Hone
Kevin Mccarthy is a principal scientist I in the cell and molecular sciences group in the department of Drug Substance Development at Wyeth Biopharma
Articles by Kevin Mccarthy
Mark Leonard is the director in the cell and molecular sciences group in the department of Drug Substance Development at Wyeth Biopharma
Articles by Mark Leonard
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