ORIGINAL PAPER Determination of genomic location and structure of the transgenes in marker-free rice-based cholera vaccine by using whole genome resequencing approach Mio Mejima • Koji Kashima • Masaharu Kuroda • Natsumi Takeyama • Shiho Kurokawa • Yoshiko Fukuyama • Hiroshi Kiyono • Kimiko Itoh • Toshiaki Mitsui • Yoshikazu Yuki Received: 30 May 2014 / Accepted: 22 July 2014 / Published online: 30 July 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract We previously developed a molecularly uniform rice-based oral cholera vaccine (MucoRice-CTB) by using an overexpression system for modified cholera toxin B-subunit, CTB (N4Q) with RNAi to suppress production of the major rice endogenous storage proteins. To establish MucoRice- CTB for human use, here we developed hygromycin phos- photransferase selection marker-free MucoRice-CTB by using two different Agrobacterium tumefaciens, each carrying a distinct T-DNA for co-transformation. In the marker-free candidates from co-transformants by segregation in the seed progeny, we selected a line with high CTB expression, line 51A, which we advanced to the T6 generation by self-polli- nation to obtain a homozygous line without down-regulation of CTB expression. Southern blot analyses showed that three copies of the CTB gene, but not the backbone of the T-DNA binary vector, were inserted into the rice genome of Muco- Rice-CTB line 51A. By whole genome resequencing, we showed that the transgenes in this line were inserted into intergenic regions in chromosome 3 and chromosome 12. We determined that two full-length copies, each containing the CTB and RNAi expression cassettes, were inserted in a tan- dem reverted orientation into chromosome 3. An additional copy of the CTB over-expression cassette with a truncated RNAi cassette was inserted into chromosome 12. These findings provide useful information for the establishment of a seed bank of marker-free MucoRice-CTB for human use. Keywords Selection marker-free  Gene location  MucoRice-CTB  Oral vaccine  Oryza sativa  Cholera Abbreviations Ab Antibody CT Cholera toxin CTB Cholera toxin B-subunit ELISA Enzyme-linked immunosorbent assay MS Mass spectrometry PBS Phosphate-buffered saline PCR Polymerase chain reaction RNAi RNA interference SDS-PAGE SDS-polyacrylamide gel electrophoresis T-DNA Transfer DNA TAIL-PCR Thermal asymmetric interlaced polymerase chain reaction WT Wild type Introduction The rice-based vaccine, MucoRice, has attracted interest as a vaccine production, storage, and delivery system for Electronic supplementary material The online version of this article (doi:10.1007/s11240-014-0575-4) contains supplementary material, which is available to authorized users. M. Mejima  K. Kashima  N. Takeyama  S. Kurokawa  Y. Fukuyama  H. Kiyono  Y. Yuki (&) Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan e-mail: yukiy@ims.u-tokyo.ac.jp M. Mejima  K. Itoh  T. Mitsui Graduate School of Science and Technology, Niigata University, Niigata, Japan M. Kuroda Crop Development Division, NARO Agriculture Research Center, Jouetsu, Japan H. Kiyono  Y. Yuki International Research and Development Center for Mucosal Vaccine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan 123 Plant Cell Tiss Organ Cult (2015) 120:35–48 DOI 10.1007/s11240-014-0575-4