I want to test for integration of a transgene and potentially determine where the transgene is integrating. Does anyone with experience in this area have any suggestions for doing this (i.e. TAIL-PCR, inverse PCR, ligation-mediated PCR, Southern blot, Target Capture Sequencing, etc.) that has worked for them?
Thank you Ali for your answer.
We have been using pcr and rt-pcr and found good results in the parental generation but are having difficulty with the transgene being maintain in later generations. Thus we want to figure out if it is stability being integrated in order to try to figure out why it is being lost over generations. Any suggestions?
Best Regards
so far the best is by using TLA (targeted locus amplification). TLA Technology uniquely enables the efficient targeted complete NGS of transgenes and their integration sties. TLA is therefore a powerful tool to characterize transgenic cell-lines and animal models and select those in which the transgene has integrated in the appropriate manner and without unintended sequence changes.
TLA analyses can determine:The genomic position/s of integration site/s; The number of integration sites; Single Nucleotide Variants in the transgene sequence; Structural changes in the transgene sequence; Sequence changes resulting from targeted transgene editing.
Good luck!
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