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The European Arabidopsis Stock Centre

PIPLine Marker

Donated by

  • Yvon Jaillais Equipe Signalisation Cellulaire (SICE), UMR 5667 Laboratoire Reproduction et Développement des Plantes, Ecole Normale Supérieure de Lyon

Click here to view all 42 of these lines.

Description

PIP Line Marker Collection

PIPlines seed

The PIPline is a multi-color and multi-affinity marker set (N2105604) that highlights phosphatidylinositol phosphate (PIP) associated with membranes in Arabidopsis thaliana. The set provides an easy way to screen for phosphoinositide subcellular localization patterns, provides stable, non-toxic expression in transgenic plants as well as biosensors with various relative affinities for each phosphoinositide. The constructs are under pUBQ10 promoter.

Nomenclature

The marker lines are available in three colors: Cyan (2xCyPET), Yellow (mCITRINE) and Red (2xmCherry). The nomenclature of the seed lines is, for example: P (for PIPline) 18C (C for Cyan), P18Y (Y for Yellow) or P18R (R for Red), the last letter indicating the different fluorophores.

Growth requirements in plants
  • all the plants with mCITRINE (YFP) are Basta resistant (all the Y lines)
  • all the plants with mCherry are Hygromycin resistant (all the R lines)
  • all the plants with CyPET are Kanamycin resistant (all the C lines)

Distribution
These seed stocks can all be ordered individually or as complete set of 22 lines (N2105604).
Individuals producing these lines

Mathilde Laetitia Audrey Simon, Matthieu Pierre Platre, Sonia Assil, Ringo van Wijk, William Yawei Chen, Joanne Chory, Marlene Dreux, Teun Munnik and Yvon Jaillais,

Plant transformation

Each PIPline construct was transformed into C58 GV3101 Agrobacterium strain and selected on YEB media (5g/L beef extract; 1g/L yeast extract; 5g/L peptone; 5g/L sucrose; 15g/L bactoagar; pH 7.2) supplemented with antibiotics (Spectynomicin, Gentamycin). After two days of growth at 28°C, bacteria were collected using a single-use cell scraper, re-suspended in about 200mL of transformation buffer (10mM MgCl2; 5% sucrose; 0.25% silweet) and plants were transformed by dipping.

PIP lines selection and confirmation

Primary transformants (T1) were selected in vitro on the appropriate antibiotic/herbicide (glufosinate for CITRINE-tagged PIPline, hygromycin for CHERRY-tagged PIPline and kanamycin for CyPET-tagged PIPline). 24 independent T1s were selected for each PIPline. In the T2 generation at least 3 independent transgenic lines were selected using the following criteria when possible: i) good expression level in the root for detection by confocal microscopy, ii) uniform expression pattern, iii) single insertion line (1 sensitive to 3 resistant segregation ratio) and, iv) line with no obvious abnormal developmental phenotypes. PIPlines for which we could not find fluorescence out of 24 independent lines or that did not associated with any membrane compartments (including the PM) were not further analysed. The remaining PIPlines were rescreened in T3 using similar criteria as in T2 with the exception that we selected homozygous lines (100% resistant). At this step, we selected one transgenic line for each PIPline that was used for further analysis and crosses and that was distributed to the Arabidopsis stock centres.

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