Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation.

BMC Biotechnology
Mohamed FaizeLorenzo Burgos

Abstract

The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently. We show here that chimeras are a very frequent phenomenon observed after regenerating transgenic plants. Spatial and temporal analyses of transformed tobacco and apricot plants with a quantitative, real-time PCR amplification of the neomycin phosphotransferase (nptII) transgene as well as of an internal control (beta-actin), used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the nptII transgene differed greatly along with the sub-cultivation period of these plants and was dependent on the localisation of the analysed leaves; being higher in roots and b...Continue Reading

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Citations

Jun 21, 2011·Yi chuan = Hereditas·Yan WangLi-Ping Chen
Nov 15, 2011·Biotechnology Advances·Biao WangQiaochun Wang
Dec 26, 2012·Gene·Shweta Mehrotra, Vinod Goyal
Aug 7, 2016·Frontiers in Plant Science·Nathaniel M ButlerDavid S Douches
May 14, 2020·BMC Biotechnology·Zsófia BánfalviMihály Kondrák
Mar 12, 2020·Physiology and Molecular Biology of Plants : an International Journal of Functional Plant Biology·Amanpreet KaurAnil Kumar
Dec 13, 2019·Royal Society Open Science·Shengxing LiHanyao Zhang

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Methods Mentioned

BETA
transgenic
PCR
X-ray

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