Location: Bioproducts ResearchTitle: Use of Quantitative PCR for Determining Copy Numbers of Transgenes in Lesquerella fendleri) Author
Submitted to: American Journal of Agricultural and Biological Science
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/5/2010
Publication Date: 12/1/2010
Citation: Chen, G.Q., Lin, J.T. 2010. Use of Quantitative PCR for Determining Copy Numbers of Transgenes in Lesquerella fendleri. American Journal of Agricultural and Biological Science. 5(3):415-421. Interpretive Summary: Lesquerella fendleri, being developed as a new industrial oilseed crop in the southwestern region of U.S., is valued for its unusual hydroxy fatty acid (HFA). One of the efforts is to increase the HFA content in seed through genetic engineering. When new transgenic plants are obtained, an essential step is to determine the copy number in the transgenic plants, because the copy number can greatly influence the expression level and genetic stability of the target gene. This manuscript describes a qPCR method that can be implemented as a tool for rapid and accurate determination of transgene copy number in L. fendleri. The results indicate a high percentage of insert rearrangement. Because the required sample size for the qPCR method is very small, the transgene copy number can be determined while primary transgenics are still in the tissue culture stage. This allows the selection of desirable transgenic plants and saves greenhouse space, which increases the capacity of the transgenic event production pipeline.
Technical Abstract: The first successful attempt to apply a real-time polymerase chain reaction (PCR)-based method to determine transgene copy number in Lesquerella fendleri is described. The system utilized a known one copy gene, LfKCS4/5, from L. fendleri as an endogenous calibrator and the threshold crossing point (Ct) measured by Applied Biosystem 7500 system to calculate the copy number of primary transgenic lines (T0). Our data demonstrated unambiguous 2-fold discrimination of copy number of ß-glucuronidase gene (gusA) and hygromycine phosphotransferase II (hptII) genes in 12 primary transgenic lines. Most of the lines contained one or two copies of each gene. Eight out of 12 samples (66.7%) showed more copies of gusA gene than that of hptII gene, suggesting rearrangements of the transferred (T)-DNA. Possible modifications of the T-DNA cassette in L. fendleri are discussed based on main models of T-DNA integration in the plant genome.