Expression plasmids for use in Xenopus

Plasmids, both vectors and controls, for the ectopic expression of gene products in developing Xenopus embryos.

If you have a useful map, or additional details on any of those presented here (including on who made the different plasmids...), please submit it to Peter.

Control plasmids and lineage tracers.

Expression vectors.

For mRNA production

pSP64T
pSP64TS, an excellent, simple, uncomplicated plasmid containing globin flanking sequences, a small polylinker and stop codons in all three ORF's. Made by Paul Wilson.
pT7TS, T7 polymerase version of pSP64TS
CS2 family of plasmids made by Dave Turner and Ralph Rupp, including myc and NLS tagged versions (Under construction!!)

For plasmid driven expression in the embryo

pCS
pCSKA
pXeX
pcDNA1

Controls.

Injected RNA and DNA is toxic to the embryo at high doses. Standard nucleic acid toxicity phenotypes include gastrulation defects (ring embryos, sometimes called the spina bifida phenotype), kinked embryos (abnormal somitogenesis), anterio-dorsal deficiencies (small heads or eyes, lack of pigment cells) and death. Nucleic acids become toxic at about 100 ug/ml, but will also be toxic at much lower concentrations if too great a volume is injected. For more details on this subject see Vize, Melton, Hemmati-Brivanlou and Harland (1993). Assays for gene function in developing Xenopus embryos, in Methods in Cell Biology, vol. 36. B.K.Kay and H.B.Peng eds. Academic Press Inc., Florida.
In addition to nucleic acid toxicity protein toxicity must also be considered. Creating a frame-shift or other mutation that blocks protein production will control only for nucleic acid, but not protein toxicity. Altering a crucial amino acid in a DNA recognition motif will not allow the non-specific effects of DNA binding activity to be evaluated as all binding will be obliterated. Altering cysteines is a good control in secreted proteins, but it is difficult to create the perfect control for any protein, mutations that effect specific function may also effect non-specific functions. If one of the common artifacts described above is observed in response to your gene product try using lower doses, injection at different times and in different places, or an alternative method of expression (e.g. plasmid).
Information provided by Peter Vize, Paul Krieg, Mike Klymkowski and Enrique Amaya.

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