12.2
Transient overexpression
(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of plasmids was estimated to be about 90% (Hiwatashi, unpublished).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)