Date of Graduation

12-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Srivastava, Vibha

Committee Member

Stewart, James M.

Second Committee Member

Murphy, J. Brad

Third Committee Member

Henry, Ralph L.

Fourth Committee Member

Sagers, Cynthia L.

Keywords

Biological sciences; Cre-lox; Flp-frt; Gene dosage; Marker gene removal; Rice transformation; Site-specific recombination; Transgene expression

Abstract

Site Specific Recombination systems, such as FLP-FRT and Cre-lox, have been successfully used for site-specific gene integration and marker-gene deletion in plant systems. They are very useful tools in the integration of single-copy full-length transgene cassettes into the genome because the transgene integration via conventional methods often generate multi-copy locus. Such complex locus containing direct and inverted repeats of full-length and truncated copies of the transgene cassette generate aberrant RNA resulting in gene silencing. Therefore, for stable gene expression, a single copy transgene locus is preferred. However, even single copy locus sometimes succumbs to gene silencing. Although the mechanism is not very well understood, it is thought that transgene expression above a threshold level triggers gene silencing. Therefore, it is important to study the effect of transgene copy number on gene expression, and to control the locus structure and integrate full-length copies. In the present study, Cre-lox site-specific recombination system was used for integration of 1 - 3 C of green fluorescent protein (GFP) or beta-glucuronidase (GUS) genes into a pre-determined integration locus in rice cells. Expression analyses revealed a clear 2 - 4 times increase in GFP and GUS productions correlated with transgene copy numbers (1 - 3C).

As a next step towards the practical implementation of this technology, a molecular strategy was developed for generating marker-free site-specific gene integration. This strategy relies on Cre-lox-mediated gene integration followed by FLP-FRT-mediated marker excision. The molecular strategy is designed to generate an integration locus consisting of strategically-placed FRT sites to remove marker genes. In the original strategy, an inducible FLP-FRT system was included to control the marker excision step. This strategy was tested on two integration lines resulting in poor to undetectable excision of marker genes. In the subsequent modifications involving re-transformation of the integration lines with the improved version of FLP gene, called FLPe, marker excision was detected in the retransformed lines. The excision footprint was detected by PCR and Southern analysis in most of the lines, and excision efficiency determined in the selected two lines by real time PCR as 75 and 100%.

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