A
gain-of-function and over-expression experiment with an inducible expression
system is useful to characterize gene functions. An induction system using a
heat shock promoter has been established in Physcomitrella (Saidi et al. 2005). We modified the system and constructed new
vectors with GATEWAY-compatibility. Furthermore, we used a neutral targeting
site, PIG1 for a reproducible integration (Okano et al. 2009).
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.
(1) pPGX6, pPGX8
The vector, pPGX6 and pPGX8 harbors
GATEWAY cassette fused to lexA operator and minimal 35S promoter, XVE chimera
receptor protein driven by GX6 and GX8 promoters, respectively. There are
originated from pER8 (Zuo et al. 2000). To select transgenic lines, the aph4
cassette for hygromycin selection is included. Both ends of that region are
linked to DNA sequences of the PIG1 targeting site as homologous regions. The
PIG1 site is an ideal neutral integration site whose targeting rates, promoter
activity, and effects to phenotype were examined in our laboratory. Excision of
the targeting DNA fragment from a back-bone vector with Sse8387I or PmeI is
necessary for efficient targeting.
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 pPGX8 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).
PIG1bR and
PIG1bL are P. patens genomic sequence
for PIG1 site, a potential neutral site (Okano et al. 2009). rfcA and 35SPA are
respectively reading frame cassette A for Gateway system and CaMv35S terminator
including poly adenylation site.
Your gene and DNA fragment are
subcloned into pENTR/D/TOPO vector by PCR. Your gene or DNA fragments in these
entry clones are integrated into the destination vector pPGX6 or pPGX8, by the
LR reaction. For details of the GATEWAY system (Invitrogen), you can refer to
websites as follows:
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html
See Chapter 11.3
To
sufficiently induce gene expression, transgenic P. patens lines harboring XVE system are soaked in 1µM
estrogen (beta-estradiol) solution.
Beta-estradiol
(WAKO, Osaka, Japan)
Stock
solution: 10 mM dissolved in DMSO, subdivide into small aliquots, and stored at
-20C.
Estradiol
concentration:
Responsibility
to beta-estradiol differs among transgenic lines and we need to examine the
amount of induction for each line with RT-PCR. If GFP gene is fused with an
induce gene, GFP signal will be a spatiotemporal marker for the induction.
Usually, beta-estradiol is used at 0.01 to 1 µM final
concentration. With the addition of estradiol at higher concentration than
0.1 µM estradiol, gametophore cells locating along the leaf edge become
malformed.
Example
1 by Minoru Kubo
7
ml of 1 µM estradiol diluted in BCDAT was poured onto BCDAT medium
harboring protonemata and gametophores. The amount of induction was examined
with RT-PCR. A targeted gene is induced 50 times after 3 hours and 100 times
after 6 hours. It becomes mostly stable after 48 hours. We do not know how long
the induction continues.
Example
2 by Akitomo Nagashima
25
ml 0.1 µM (or 0.2 µM) estradiol diluted in MES (pH6.5, 10mM) was
poured onto BCDAT medium harboring protonemata and gametophores. The
protonemata and gametophores were cultured for 36 hours. Leaves from the
gametophores were excised and put into MES (pH6.5, 10mM) with 0.1 µM
estradiol at final concentration to observe reprogramming of a leaf cell to a
chloronema apical cell
Example
3 by Chaoyang Cheng
Phenotype
analysis of genes regulating gametophore induction: Cultured protonemata
on BCDAT medium supplemented with a final concentration 2 µM estradiol
for 3 weeks and observed gametophore phenotype.
Example
4 by Chaoyang Cheng
Phenotype
analysis of reprogramming of an excised leaf cell into a chloronema apical
cell: Gametophores were cultured on BCDAT medium. The gametophores were
moved into MES (pH6.5, 10mM) with 0.2 µM estradiol and
incubated for 2 days at 25C. The gametophore leaves were excised and the
reprogramming was observed in MES with 0.1 µM estradiol.
Example
5 by Masaki Ishikawa (Ishikawa et al. in prep.)
To
keep the induction of gene expression:
1.
Culture protonemata and gametophores for several weeks on a regular medium (BCD
or BCDAT).
2.
Collect gametophores and soak into BCDAT medium with 1 µM beta-estradiol
(final concentration) for 24 hours.
3.
Cut gametophore leaves and soak the excised leaves in BCDAT liquid medium with
or without 1 µM beta-estradiol for 3 days.
4.
Analyze the phenotype.
Zuo
et al. (2000) An estrogen receptor-based transactivator XVE mediates highly
inducible gene expression in transgenic plants. Plant J, 24, 265-273.
(1) pPIG1HG
The
vector, pPIG1HG harbors a GATEWAY cassette driven by a soybean GmHSP17.3
promoter and the aph4 cassette for hygromycin selection of transgenic P. patens. Both ends of this construct
are flanked by sequences of the PIG1 sequence for targeting. This is an
intergenic region in the P. patens
genome as has been shown to be a good targeting site, in terms of targeting
rate, promoter activity and no observable effect on phenotype when targeted. Linearization
of pPIG1HG by Sse8387I or PmeI increases targeting efficiency.
(2) pPIG1HCG,
pPIG1HGC
The
vectors pPIG1HCG and pPIG1HGC harbour a GATEWAY cassette driven by a soybean
GmHSP17.3 promoter and the aph4 cassette for hygromycin selection of transgenic
P. patens. Both ends of this region
are flanked by sequences of the PIG1 targeting site. pPIG1HCG and pPIG1HGC are
able to make cerulean*-fusion protein at the N-terminus and C-terminus,
respectively. Transformants using these constructs are easy to monitor for gene
expression by fluorescence microscopy.
* cerulean : a variant of cyan fluorescent protein
Your
gene and DNA fragment are subcloned into pENTR/D/TOPO vector by PCR. Your gene
or DNA fragments in these entry clones are integrated into destination vectors
such as pPIG1HG, pPIG1HCG and pPIG1HGC, by the LR reaction. For details of the
GATEWAY system (Invitrogen), you can refer to websites as follows: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Gateway-Cloning.html
See Chapter 11.3
Transgenic
P. patens harboring HSP system are
cultured at 25℃.
For heat-shock treatment, they are transferred to 38℃ for 1hr and then returned to 25℃. Repeated heat-shock
cycles (e.g. 38℃
for 1hr: 25℃
for 11hr) are beneficial in some instances.
Okano, Y. et al. (2009) A polycomb repressive complex 2 gene regulates
apogamy and gives evolutionary insights into early land plant evolution. PNAS, 106,
16321-3
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.