|Sangtong, Varaporn - ISU|
|Long, Mary - ISU|
|Lee, Michael - ISU|
Submitted to: Plant Molecular Biology Reporter
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 28, 2001
Publication Date: June 11, 2001
Citation: SANGTONG, V., MOTTL, E.C., LONG, M.J., LEE, M., SCOTT, M.P. SERIAL EXTRACTION OF ENDOSPERM DRILLINGS (SEED)-A METHOD FOR DETECTING TRANSGENES AND PROTEINS IN SINGLE VIABLE MAIZE KERNELS. PLANT MOLECULAR BIOLOGY REPORTER. 2001. v. 19. p. 151-158. Interpretive Summary: Transgenic plants are used by scientists as tools to investigate many aspects of plant biology. Scientists working with transgenic plants normally characterize several aspects of expression of the transgene in large numbers of plants in order to evaluate the impact of the transgene. This is often a laborious process. This paper describes an efficient method for characterizing the expression of transgenes in large numbers of maize kernels. A single tissue sample is subjected to several types of analyses, minimizing the effort required to sample tissue. This method has allowed us to characterize expression of the wheat 1Dx5 HMW glutenin in transgenic maize kernels. This method will allow scientists working with transgenic corn to analyze their transgenic seed more efficiently.
Technical Abstract: We have developed a method for detecting a transgene and its protein product in maize endosperm that allows the kernel to be germinated after analysis. This method should have broad utility in several monocots and dicots. Our method involves first sampling the endosperm with a hand-held rotary grinder so that the embryo is preserved and capable of germination. This tissue is then serially extracted, first with SDS-PAGE sample buffer to extract proteins, then with an aqueous buffer to extract DNA. The product of the transgene can be detected in the first extract by SDS-PAGE with visualization by total protein staining or immuno-blot detection. The second extract can be purified and used as template DNA in PCR reactions to detect the transgene. This method is particularly useful for screening transgenic kernels in breeding experiments and to test for gene silencing in kernels.