Research Experience in Molecular Biotechnology & Genomics Summer 2008. Center for Integrated Animal Genomics. Regina S. Nickel, Toya Lawrence, Becky Weeks, Erik Vollbrecht. Using the Ac-Ds transposon system in maize as a tool for forward mutagenesis. Abstract. Objectives. Results.
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Center for Integrated Animal Genomics
Regina S. Nickel, Toya Lawrence, Becky Weeks, Erik Vollbrecht
Using the Ac-Ds transposon system in maize as a tool for forward mutagenesis
We were able to develop assays for PCR for each gene of interest to test for insertions of Ds.
The goal of this project is to determine if a Ds transposon can be used for local mutagenesis. Lines were chosen that possessed Ds insertions near genes of interest. These lines were genotyped using a special PCR assay and tagged for outcrossing to initiate remobilization. We will screen the progeny of these crosses to determine if Ds elements have inserted into the designated genes. If successful, researchers will be able to search
Table 1 Subset of lines genotyped for Ds insertions.
Materials and Methods
Novel insertion site
Figure 1. Mobilization of Ds transposons affect kernel color
Digest cuts here
Digest cuts here
Figure 3. Orientation of gene specific primers and Ds primers relative to transposon and insertion site.
Conrad, L.J. and Brutnell, T.P.(2005). Ac-Immobilized, a stable source of Activator transposase that mediates sporophytic and gametophytic exision of Dissociation elements in Maize. Genetics 171. 1999-2012.
McCarty, D.R., Settles, A.M., Tan, B.C., Latshaw, S., Porch, T., Robin, K., Bairer, J., Avigne, W., Lai, J. et. al (2005) Steady-state Transposon Mutagenesis in inbred Maize. Plant J. 44, 52-61.
A special thanks to Erik Vollbrecht for hosting me in his lab and to the members of the Vollbrecht lab for their technical help and advice throughout the project.
I would also like to thank Dr. Max Rothschild for his guidance and for giving me the opportunity to participate in the REU Summer Internship program.
Last, I would like to thank Linda Wild for working so hard to get me into the REU Summer Internship program.
Figure 2 IPCR schematic. Picture modified from figure by Justin Schares
Bands Confirming Ds Insertions
But what if a Ds element is located close to, but not in a gene of interest? Can it be used for local mutagenesis? In other words, can it be mobilized into the nearby gene?
Figure 4. Example of PCR confirming Ds insertion.
Program supported by the National Science Foundation Research Experience for Undergraduates