Green plasmids


Table of Methods
Plakoglobin-GFP* in living Xenopus

We have made a series of plasmids based on the pCS2-mt plasmid developed by Turner, Rupp & Weintraub. We mutated the GFP moeity to increase its efficiency, as described by Heim & Tsien (1996. Current Biology 6:178-182).

Both the Ser65 Thr and the Ser65 Ala mutations appear to be equally efficient in our experience in both Xenopus and mammalian cell systems.

The pCS2 plasmid can be used to synthesize RNA via its SP6 promoter, or directly drive expression in eukarotic cells via the CMV promoter. When pCS2-GREEN plasmid DNA is injected directly into the nuclei of cultured Xenopus A6 or rat kanagaroo PtK1 cells, green fluorescence can be easily visualized within 1 to 2 hours (using standard fluorescein optics).
 

RNA encoding GFP can be seen in Xenopus embryos for many days and appears to be non-toxic. We have made a number of different versions of the pCS2 plasmid, and we are willing to supply them to you if you are interested -- just contact us.

Transplant of neural crest from GFP RNA injected embryo into a uninjected embryo.
In pCS2mt-GFP, the S »T mutant form of GFP has been inserted  such that the myc tags and the GFP are in-frame.   

Alternatively, you can subclone your favorite sequence  between the myc tags and the GFP using EcoRI and Xba I sites.

This produces an N-terminally myc-tagged, C-terminally GFP-tagged  chimeric polypeptide.

The EcoRI site's AAT defines the reading frame for the insert.

We are not always sure which version of pCS2mt-GFP* we are sending (i.e. with or without the BamHI site), so please check if you care! 

We also have a version of this plasmid in which the myc-tags have been removed; this is when we are co-injecting RNAs encoding a myc-tagged polypeptide.

To place a single myc-GFP tag at the N-terminus of your polypeptide, we use pCS2mycGFP.  In these plasmids, subcloning into the EcoR/Xba I sites leads to the expression of a chimeric polypeptide with an N-terminal, single myc-GFP tag. 

The ~28kDa GFP moiety can influence the behavior of the chimeric polypeptide, so it is probably a smart move to make both N- and C- GFP tagged forms of your polypeptide of interest.

We send out the pCS2mycGFP* plasmid containing the XTCF3 or Slug sequence for reasons to complex to discuss here!

Remove the insert with a EcoRI / Xba I digest.

We purchased the pQBI50 plasmid from Quantum BioTechnologies Inc and then used PCR to move the BFP moeity into pCS2 to create pCS2mt-BFP.  However, the blue fluorescence is not very strong, we do not send it out.  There is probably a better version available now, and we would not want to disappoint anyone! 

blue plakoglobin in the nucleus of Xenopus A6 cells

When using a blue plasmid we recommend the protocol of Rizzuto et al (1996. Curr. Biol. 6:183-188); add 10µM Troxol (a soluble vitamin E analog --from a 10mM stock in DMSO) to the culture media to minimize bleaching.  

Addition hints:

Visualization: We use Zeiss IM35 invert microscopes and a 75W Xenon light source for standard visualization (and fancy schmancy confocal and deconvolution microscopy for other stuff). Standard FITC filters work fine for GFP, filters for BFP can be purchased from Omega Optical (802)-254-2690.

Take care, looking at GFP too long leads to increased red autofluorescence, which can cause experimental confusion. After 24 hours, the GFPs can easily been seen using a 2.5X lens! (probably expression is a little too robust).
Table of Methods
  
revised 3 August 2002