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0:00
My name is Christof von Kalle
from the National Center for Tumor
Diseases in Heidelberg in the
German Cancer Research Center.
I'm going to be talking about
tracking vector insertion sites
to explore the biology of
transduced cells in vivo.
0:16
Integrating vectors have been
used for a number of years
for the genetic
modification of cells.
This has been particularly
done extensively
in that formation, hematopoiesis.
Integrating vectors
have the properties
to place a copy of the
profile DNA somewhere
in the genome of
the transfused sell.
This integration occurs
depending on the vector system
in a semi-random
fashion and is stable.
You can see that a marking that
occurs in a progenitor or a stem
cell is actually then passed
on to the progeny of that cell,
meaning that sampling of the
peripheral blood and analysis
for the integration sites
can delineate the activity
of progenitor and stem cells
in terms of their contribution
to different blood
lineages, the numbers
or cells, and their
activity over time.
1:17
The next slide shows the
possibility to analyze
such insertions in
very complex mixtures.
Usually if the blood formation
of an animal or a patient
is marked in part by retro
viral or anti-viral vectors,
the analytes that you need to find
this composition in a very complex
majority of cells may
not be transfused.
The number of cells or a cell
clones that carry a genetic vector
may be very large, and so in terms
of the analyte to be analyzed,
this can be very complex mixtures.
So a number of years ago,
we have revised methodology
that uses the unique
abilities of polymerases
to create transcripts of
such insertional events.
We used linear PCR
going from one primer
out into the genome to create
a high number of copies
of every insertional events in the
given sample or analyte which gives
us the opportunity to
then enrich these and go
through different steps
of enrichment, ligation,
and amplification without
losing at least all
of the copies of a given event.
So that the integration site
analysis that we then can perform
is very sensitive for the
presence of each individual event
just by the very fact that we have
been able to create multiple copies
of each individual event
at the very beginning
of the steps of this method.
The restriction length polymorphism
that is created by a such reaction
shows multiple bands, so one can
see multiple, amplified fragments
of different size indicating
that LTR insertion
loci with different DNA lengths
have been amplified by this method.
