Measurement of transgene copy number in transgenic tobacco plants producing human interferon-γ (hIFN-γ) using quantitative real-time PCR

Abstract

In transgenic plants, the transgene copy numbers can highly affect the level of expression and genetic stability of the transgene. Hence, the first step in their characterization is the estimation of transgene copy numbers integrated in the plant genome. Quantitative real-time PCR (qRT-PCR) was used to determine the copy numbers of human interferon-γ (hIFN-γ) and hygromycin phosphortransferase II (hptII) transgenes in the genome of the T₀ generation of 18 transgenic tobacco lines using the axi1 gene as an endogenous control. With optimized PCR conditions, we attained highly exact estimates of one, two, three, and/or four transgene copies in the T₀ transformants. Moreover, estimation of copy numbers of the hIFN-γ transgene and the hptII selective marker gene indicated that rearrangements of the T-DNA has regularly happened in transgenic tobacco. Transgene copy number was also estimated using Southern blot analysis of gDNA derived from transformants. The transcript level and expression amount of recombinant hIFN-γ protein were evaluated in various events using RT-PCR and enzyme-linked immunosorbent assay (ELISA) techniques. A disagreement between the transcript level and the amount of recombinant protein with an inverse correlation between transgene copy number and expression level observed in some events, probably showing translational gene silencing and co-suppression or silencing, respectively. These results were also compared with segregation ratios of hygromycin-resistant phenotype in T₁ plants of each line and found to be, in general, consistent.