9.3  Transient overexpression

Tomomichi Fujita


(1) pTFH22.4

The vector, pTFH22.4 harbours a GFP expression cassette together with a cDNA expression cassette driven by a strong rice actin promoter*.  Due to this GFP cassette, it is possible to distinguish transformed cells from untransformed cells by GFP fluorescence.  GFP fluorescence can be detected in regenerated protonemata with 10-cells, even though this plasmid is ,in most instances, not integrated in a nucleus.  When you culture the transformants on G418 selection plates, the GFP signal remains visible even in protonemata with more than 20-cells and in gametophores.


* rice actin reference



(2) pTKM1, pTKM1sGFP, pTKM1DsRED

pTKM1 is an overexpression vector similar to pTFH22.4, but lacks an nptII-GFP cassette. This plasmid has an advantage over pTFH22.4 because of its smaller size. To select transformed cells, you need to co-transform this plasmid with pTKM1sGFP or pTKM1DsRED.  The rate of co-transformation with two kinds of plasmids was estimated to be about 90% (Hiwatashi, unpublished).


(3) pUGW0

This vector contains the  CaMV35S promoter for constitutive cDNA expression and a gateway recombination cassette to introduce your cDNA. If you are used to the gateway system, it's easy to construct a transient overexpression plasmid. The 35S promoter is much weaker than the rice actin promoter. 

pUGW0 was kindly provided by Dr. Tsuyoshi Nakagawa (Shimane University)