Yfg

(►) Recombinase target site

(►) Recombinase target site

Three possible schemes of making target constructs for mosaic analysis. Other configurations and orientations are possible. (Triangles in circles) Recombinase target sites; (arrows) direction of transcription. The top line in each panel shows the configuration before recombination, and the bottom line shows the configuration after recombination. (YFG) Your Favorite Gene; {P3SS) CaMV 35S promoter; (Pypr) YFG promoter; (NOS) nos terminator.

• In the first scenario (Figure 8.3A; see also Sieburth et al. 1998), the target construct contains a genomic clone of your favorite gene, YFG, that can rescue the mutant phenotype of interest and a constitutively expressed reporter gene (35S::GUS) positioned between two recombinase sites. This target construct is introduced into a mutant background and single-insert lines with a completely rescued phenotype are selected. The presence of a single target insert is crucial, because multiple targets will obscure a sector if one target has recombined and another has not. Next, the recombinase source in a mutant background is crossed to the rescued mutant containing a single-insert target. The progeny of this cross should then be homozygous for the mutation of interest and hemizygous for both the recombinase source and the target transgene. These plants are then heat-shocked (for an empirically determined length of time) at an early stage of development and then scored later for mutant sectors (which will be white after X-Gluc staining, or GUS-negative). Cell-autonomous behavior is indicated if all white tissue is phenotypically mutant and all blue tissue is phenotypically w::d type.

• The second scenario (Figure 8.3B) is similar to the first, except that mutant sectors will be blue in a white plant. A transcriptional terminator is placed after the first recombinase site to prevent read-through from the CaMV 35S promoter into YFG. It might be advantageous to have the marker and YFG in opposite orientations, instead of tandem orientation as shown in the figure.

• The third arrangement (Figure 8.3C) generates gain-of-function sectors, which will be white in a blue plant (Riou-Khamlichi et al. 1999). Such experiments can be performed in wild-type or mutant backgrounds and can be used in cases where constitutive expression in all cells of the plant is detrimental or to rescue mutant cells (Sessions et al. 2000).

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