9.3 Transient overexpression
(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(Hiwatashi, unpublished).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)