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Tracking vector insertion sites to explore the biology of transduced cells in vivo

Published on September 3, 2014   35 min

A selection of talks on Genetics & Epigenetics

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.
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Tracking vector insertion sites to explore the biology of transduced cells in vivo

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