Monday, June 12, 2017

Last update

T-DNA Ds* launching platforms

 

More than 200 Agrobacterium-transformed lines carrying a transpositionally active marked Ds* element have been generated and the location of 82 active platforms has been mapped to date (see below).  The development and use of this novel transgenic reverse genetics resource is outlined in the ensuing paragraphs.

Since both parents of the Hi-II hybrid used for transformation are c1 mutants, we based our transposon system on the c1-m2 allele, which carries a Ds element in the third exon.  Ac1 wx-m7(Ac); R line was derived to assay mobility of the engineered Ds* transposons in Hi-II (which is c1r).  An advantage of a c1-based selection system is that most inbreds are c1, so researchers can cross the c1-m wx-m7(Ac) stocks directly to theirs in order to generate Ds*-tagged mutations of their gene of interest.

TAG21 is a pTF102-derived binary vector based on c1-m2.  The Ds* element carries a construct in which an alpha-zein promoter drives expression of GFP (green fluorescent protein).  P(zein)/GFP conditions green fluorescent endosperms under blue light.  Germinal transpositions of Ds* are easily identified in single-copy transgenotes as revertant purple kernels (C’) that fluoresce green (i.e., retain Ds/GFP).

A Ds-based transgenic system

 

Most transgenic lines carry a single copy of the T-DNA.  However, some carry more than one, so the number of T-DNA platforms in our active transgenic lines is close to 100 .  So far, 82 TAG21 platforms have been mapped on the B73 reference genome, as shown in the figure below.

Chromosome location of the T-DNA launching pads

Over 3000 C’ green fluorescent revertant kernels have been isolated from platforms A6A on 1L and A20F on 9L and 8000 C’ from A20BB on 6L.  All these individuals carry  trDs* elements.  The site of insertion of 1823 trDs* elements (dsg sites), both linked and unlinked to their respective donor platforms, has been determined by high throughput sequencing of 3-D transposant pools and added to our website under "DATA RELEASED:  New sequence-indexed Ds* sites by NGS of 3-D transposant pools ”.  Seed from many sequence-indexed dsg  lines are available from the Maize Stock Center and all of them will be eventually.  Lines that are not can be obtained directly from the Dooner lab at this time.