11.5
Dominant repression by chimeric repressor silencing technology (CRES-T)
Minoru Kubo, Yasuko Oguri,
and Mitsuyasu Hasebe
Introduction
Induction of a dominant negative form of protein is useful to
infer functions of genes with redundancy. In the chimeric repressor silencing
technology (CRES-T), a transcription activator fused to SRDX, an artificial
peptide containing an EAR-repression motif is effectively used for Arabidopsis (Hiratsu et al.,
2003). To apply CRES-T for P. patens,
we developed several vectors. SRDX-fused protein may cause lethality and growth
defect and we employed inducible systems by estrogen or heat shock. We
recently found problems on the heat shock promoter system in the following
points: (1) the heat shock promoter tends to be silenced after cultivation for
several weeks under regular culture conditions at 25°C and we have to inoculate
transgenic lines each time from a stock at 4°C; (2) continuous induction at
37°C enhances the silencing; and (3) heat shock at 37°C for more than a week
causes growth and developmental defects. Thus, we constructed vectors harboring
estrogen-inducible system with the XVE chimera receptor protein (Zuo et al.
2000), which enables us more than 100 times of gene expression and to keep the
expression level for at least 96 hours with continuous estrogen supply.
11.5.1 CRES-T with XVE (estrogen)-inducible
expression system
Minoru Kubo, Yasuko Oguri,
Kari Thompson, Kaori Miyawaki, Akihiro Imai, Tetsuya Kurata, and Mitsuyasu
Hasebe
Materials and Methods
pPGX6DR and pPGX8DR
Each destination vector pPGX6DR and
pPGX8DR harbors a lexA operator, a minimal 35S promoter, a GATEWAY cassette, a
SRDX peptide coding necleotides, and a XVE chimera
receptor-coding gene driven by a GX6 and a GX8 promoter, respectively. This DNA
fragment will be inserted in the PIG1 neutral targeting site (Okano et al.
2009) by homologous recombination. Complimentary DNA fragment cloned in pENTER/D/TOPO is destinated to pPGX6DR or pPGX8DR by the LR
reaction (http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html)
and the SRDX is fused to the C terminus. The GX6 promoter is a 5’ non-coding
region of a conserved hypothetical protein (BJ183999) and the GX8 promoter is
that of KINID1a (AB434497: Hiwatashi
et al. 2008). The GX6 promoter more efficiently induces an introduced gene than
the GX8 promoter does. However, the GX6 promoter does not function in
gametophore shoot apex including young leaves until 5th from the tip
and protonema apical cells. To induce a gene in whole gametophore and protonema
cells, the pPGX8DR vector is suitable. Spatial and temporal activity of GX8
promoter is examined using a control line (pPGX8 [see chapter 11.7] with a nuclear localization signal-GFP-GUS fused
gene at the integration site).
pPGX6DR
and pPGX8DR plasmids contain the aph4 cassette for hygromycin selection. Both plasmids should be linearized by Sse8387I or PmeI for efficient targeting.
How to make a construct
A DNA fragment of an induced gene
should be subcloned into the entry vector pENTR/D/TOPO,
which then is destinated to the destination vector pPGX6DR and pPGX8DR by the LR
reaction (http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html).
Transformation and selection of candidate lines
See Chapter 11.3
Inducible conditions
See
Chapter 11.7
11.5.2 CRES-T with Heat Shock Prompter-inducible
expression system
Materials and Methods
pPHGDR
The
vector, pPHGDR harbors a GATEWAY cassette driven by a
soybean GmHSP17.3 promoter (Saidi, Y., et
al. 2005) and
an aph4 cassette for hygromycin selection. This DNA fragement
is bound with genomic DNA fragements of the PIG1
targeting site for homologous recombination. PIG1 site is an intergenic region and a putative neutral site (Okano et al.
2009). Linearization of pPHGDR by Sse8387I or PmeI increases targeting efficiency. pPHGDR is a vector to fuse SRDX to the C terminus of
an induced gene.
How to make a construct
A
DNA fragment of an induced gene should be subcloned
into the entry vector pENTR/D/TOPO, which then is destinated to the destination
vector pPHGDR by the LR reaction (http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html).
Transformation and selection of candidate lines
See Chapter 11.3
Inducible condition
Transformed
P. patens is cultivated at 25℃,
shifted to 38℃ for 1hr heat shock, and returned to 25℃.
Repeats of a heat-shock cycle (38℃ for 1hr and 25℃
for 11 hr) are beneficial for mostly continuous induction (Okano et al. 2009).
References
Hiratsu, K., Matsui, K., Koyama, T., and Ohme-Takagi, M. (2003) Dominant repression of target genes
by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant J. 34: 733–739.
Okano Y,
Aono N,
Hiwatashi Y,
Murata T,
Nishiyama T,
Ishikawa T,
Kubo M,
and Hasebe M.
(2009) A polycomb repressive complex 2
gene regulates apogamy and gives evolutionary
insights into early land plant evolution. Proc. Natl. Acad. Sci. USA 106:
16321-16326.
Saidi, Y., et al. (2005) Controlled expression of recombinant proteins in Physcomitrella patens by a conditional
heat-shock promoter: a tool for plant research and biotechnology. Plant Mol.
Biol. 59: 697- 711.