Mechanistic models for modification of methylation status of targeted alleles by GT.

<p>Methylated cytosines are indicated by grey lollipops. The GT vector is denoted by a red line, with a red asterisk illustrating the genetic perturbation introduced by the vector. If gap enlargement occurs following DSB induction (pathway on the left), the methylation template is lost and the targeted allele loses its methylation. However, if GT occurs via strand assimilation (pathway on the right) or via DSB induction but without gap enlargement (pathway in the middle), the methylation pattern of the WT can be restored upon maintenance methylation.</p

Methylation changes at the targeted <i>PPOX</i> locus in lines TGT-2 and TGT-3.

<p>(<b>A</b>) The methylation landscape in TGT-2 and TGT-3 lines (upper and lower graphs, respectively). Blue circles, triangles and squares correspond to WT, T2 and T3 generations, respectively. Dotted lines mark the CG positions at which the methylation level had changed relative to WT. (<b>B</b>) CG positions that lost methylation stability in the two targeted lines. CG position is denoted beneath each bar. The numbers above each bar denote the level of methylation as fraction.</p

Loss of CG methylation at the targeted <i>PPOX</i> locus in the TGT-1 line.

<p>Cytosine methylation data obtained from bisulfite-sequencing of the PPOX fragment in the targeted line TGT-1 and two WT lines from ecotypes <i>Columbia (Col)</i> and <i>Wassilewskija</i> (Ws). Each circle represents a cytosine residue, either methylated (full circle) or non-methylated (empty circle). Cytosines are color-coded by their sequence context: red for CG, blue for CHG and green for CHH (H is C, T or A). Each row represents an independent clone. The numbers at the top of the columns indicate the position in base pairs, relative to the first base in this fragment. The second SNP introduced by the GT vector, is shown by an asterisk.</p

Ectopic Gene Targeting (EGT)-derived duplication of the <i>PROHIBITIN1</i> gene and subsequent changes in DNA methylation.

<p>(<b>A</b>) Schematic presentation of the WT <i>CRUCIFERIN3</i> locus, including the upstream <i>PROHIBITIN1</i> gene. Methylated cytosines are marked by vertical lines whose heights are proportional to the level of methylation (Cokus, Feng et al. 2008; Lister, O'Malley et al. 2008). The region analyzed by bisulfite-sequencing is highlighted in yellow. (<b>B</b>) The GT vector, containing sequences homologous to the target (dark blue) as well as the mRFP fluorescent marker (red), invades the <i>CRUCIFERIN3</i> target and primes DNA synthesis (dark blue dotted arrow), copying the <i>PROHIBITIN1</i> sequence. (<b>C</b>) and (<b>D</b>) Genetic and epigenetic consequences of EGT: (<b>C</b>) Methylation status at the endogenous copy, in T2 (upper scheme) and T3 (lower scheme) generations after GT. The red vertical line marks the newly methylated CG at position 18. (<b>D</b>) The vector including the <i>PROHIBITIN1</i> fragment that had integrated into chromosome 1 (green dotted lines) creating gene duplication. The methylation status of this duplicated copy is shown, in T2 (upper scheme) and T3 (lower scheme) generations after GT. Grey bars: the vector RB (right border) sequences.</p