11. 7 Inducible expression

Minoru Kubo and Mitsuyasu Hasebe

Introduction

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.

11.7.1 XVE (estrogen) system

(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 5^th 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.

How to make a construct

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

Transformation and selection of candidate lines

See Chapter 11.3

Inducible conditions

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.

References

Zuo et al. (2000) An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J, 24, 265-273.

11.7.2 Heat Shock Promoter system

(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

How to make a construct

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

Transformation and selection of candidate lines

See Chapter 11.3

Inducible conditions

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.

References

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.