Vibha's Research Page

 

I. Site Specific Gene Integration

 

Our laboratory is developing DNA recombination (Cre-lox and FLP-FRT) based plant transformation approaches such as site-specific gene integration and marker gene deletions.  These approaches are useful in optimizing the production of single-copy events and minizing transgene silencing.  When these approaches are used in combination with particle bombardment, single copy clones are produced at a higher rate, which express the gene-of-interest.  Thus, incidence of gene silencing is greatly reduced.  The site-specific integration approach is also useful for gene stacking as precise integrations of multiple gene copies results in proper expression of each copy.  These transformation approaches were developed using Cre-lox and FlP-FRT systems, but can be practiced with alternative recombination systems that function robustly in the plant cells.  Following transformation stratgies have been developed using particle bombardment for DNA delivery:

(i) Resolving complex multi-copy insertions to a single-copy via Cre-lox recombination.

(ii) Site-specific transgene integration via Cre-lox or FLP-FRT recombination

(iii) Marker gene deletion via Cre-lox or FLP-FRT recombination

(iv) Marker-free site-specific gene integration using FLP-FRT and Cre-lox recombination systems.

 

II. Epigenetic Mechanisms Associated With DNA Methylation in Exonic Sequences

We isolated phyA (phytochrome A gene) epi-allele, phyA', from a transgenic line of Arabidopsis containing multiple copies of PHYA transgene. phyA' istranscriptionally suppressed and highly stable in the absence of transgene locus. CG hypermethylation in exon 1 was found to be associated with its transcriptional suppression.  Further, methylation of a specific CG site rather than methylation density within the coding region was found to be critical for phyA' suppression.  Since, regulatory elements of PHYA gene are not predicted to be within the coding region, this type of gene regulation is distinct from that of promoter methylation.  Most of the well known epigenetic modifiers were ruled out to have any role in phyA' regulation; thus, transcriptional silencing mediated by exonic methylations might recruit a novel epigenetic pathway.  We are interested in understanding the underlying mechanism. 

 

       WT               phyA'            phyA-211