12.2  Transient overexpression

Tomomichi Fujita


(1) pTFH22.4

A vector, pTFH22.4 harbors GFP expression cassette together with cDNA expression cassette driven by a strong rice actin promoter*.  Due to this GFP cassette, it is reliable 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 not likely integrated in a nucleus.  When you culture the transformants on G418 selection plate, the GFP signal is 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 a nptII-GFP cassette. This plasmid has an advantage to pTFH22.4 because of its smaller size. To select transformed cells, you need to co-transform this plasmid with pTKM1sGFP or pTKM1DsRED.  A rate of co-transformation with two kinds of plasmids was estimated to be about 90% (Hiwatashi, unpublished).


(3) pUGW0

This vector contains 35S CaMV promoter for a constitutive cDNA expression and gateway recombination cassette to introduce your cDNA. If you are used to gateway system, it's easy to construct a transient overexpression plasmid. 35S promoter works more weakly than the rice actin promoter does. 

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