A novel magnetic resonance-based method to measure gene expression in living cells

In unicellular and multicellular eukaryotes, elaborate gene regulatory mechanisms facilitate a broad range of biological processes from cell division to morphological differentiation. In order to fully understand the gene regulatory networks involved in these biological processes, the spatial and temporal patterns of expression of many thousands of genes will need to be determined in real time in living organisms. Currently available techniques are not sufficient to achieve this goal; however, novel methods based on magnetic resonance (MR) imaging may be particularly useful for sensitive detection of gene expression in opaque tissues. This report describes a novel reporter gene system that monitors gene expression dynamically and quantitatively, in yeast cells, by measuring the accumulation of inorganic polyphosphate (polyP) using MR spectroscopy (MRS) or MR spectroscopic imaging (MRI). Because this system is completely non-invasive and does not require exogenous substrates, it is a powerful tool for studying gene expression in multicellular organisms, as well.


INTRODUCTION
In situ hybridization is the most precise, currently available method for obtaining spatial information on gene expression at the subcellular level. However, a major drawback of this method is that it cannot be performed in living cells or in real time: the cells or tissues to be analyzed must be immobilized and exposed to harsh chemicals that are incompatible with living cells. Thus, spatial and temporal information on gene expression cannot be measured simultaneously using in situ hybridization technology. In recent years, several noninvasive methods for detecting gene expression have been developed, which do not have this limitation. These methods use reporter genes, such as luciferase or green fluorescent protein, whose expression stimulates an optically detectable signal (1,2). Although these methods can be used in living cells, the optical signals on which the methods depend only penetrate 1-2 cm of tissue; thus, internal tissues of animal or human subjects cannot be analyzed, because the gene expression signal is obscured by light absorption and/or light scattering before it can reach the signal detector (3). Radionuclide imaging methods such as positron emission tomography (PET) and single-photon emission tomography (SPECT) offer the advantages of a non-invasive method and can measure gene expression in internal tissues (3). These techniques are well-suited for clinical use, but the spatial resolution, which is 1-2 mm, is not sufficient for research applications that demand resolution at or below the single cell level. A second disadvantage of these methods is that they require exposure of the subject to radioactive isotopes, which could be undesirable or disallowed for some applications.
MR spectroscopic imaging (MRI) is a non-invasive method with a spatial resolution at least 10 times greater than radionuclide imaging that can be used to visualize deep tissues (3). Weissleder (4) and Meade (5) were among the first to use MRI-based detection with reporter genes encoding the transferrin receptor and b-galactosidase, respectively, to analyze gene expression in cells treated with specially designed The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org substrates for these gene products. More recently, an MRIbased reporter system was developed using either the heavy subunit of ferritin or the light and heavy subunits of ferritin. This system does not require use of a specially designed substrate, but depends on the fact that ferritin is a metalloprotein that becomes superparamagnetic when it binds endogenous iron (6,7). This method, which was developed and applied in mice, may be more versatile than other non-invasive methods which require exogenous substrate. However, MRI contrast obtained by the ferritin system is limited, since the spin-lattice (7) or spin-spin (6,7) NMR relaxation rate of water proton magnetizations was reported to be typically enhanced only about a factor of two by the presence of the iron-ferritin complex, and thus the complex serves as a negative contrast agent in T 2 -and T 2 *-weighted images. The acquired MRI images may not be straight-forwardly interpreted, as the relaxation rates can be affected by other factors and may not be suitable for fully quantitative measure of gene expression.
Polyphosphate (polyP), a linear polymer of orthophosphate residues linked by high-energy phosphoanhydride bonds, is found in all organisms from bacteria to mammals (8). In yeast, most of the cellular polyP ($99%) is in the vacuole where it presumably serves as both phosphate storage and a cation chelator (8). The amount of polyP in the cell depends on the function of the vacuolar transporter chaperone (Vtc) complex (9) and the vacuolar H + -ATPase (V-ATPase) (10). Strains defective in either complex produce almost no detectable polyP, but still grow normally on acidic YPD media (data not shown) (11). These observations indicate that polyP is dispensable for growth under normal conditions and suggest that the polyP system could be manipulated without deleterious effects in living cells.
This study describes a novel MRS/MRI-based reporter system that quantifies gene expression in living cells by measuring the accumulation of inorganic polyP. This method does not require exogenous substrate and is suitable for quantitative studies of gene expression in internal tissues. In order to characterize this method, initial studies were carried out in Saccharomyces cerevisiae, because yeast accumulate polyP at a higher concentration ($120 mM) than other eukaryotes (8) and because yeast cells are amenable to genetic manipulation.

Yeast strains
Standard techniques were used for yeast growth and transformation (12). The yeast strains used in this study are listed in Supplementary Table 1. Oligonucleotide sequences used for the strain construction are listed in Supplementary Table 2. More detailed information is also available on request.
All gene replacements were confirmed by PCR, Southern blot and genomic DNA sequencing.

Northern blot analyses
Northern blot analyses were performed as described previously (15). To prepare the probes, DNA fragments surrounding the initiating methionine (VMA2 and ADH1) or corresponding to the entire open reading frame (VTC1) were amplified by PCR from yeast genomic DNA, purified and 32 P-labeled by random priming.

Immunoblot analyses
Immunoblot analyses were conducted as described previously (16). Polyclonal antibodies directed against TBP were prepared as described (16). Antibodies against the HA epitope tag (F7, mouse monoclonal) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
In vivo MR spectroscopy and MRI 31 P-NMR spectra were recorded on a DRX-500 spectrometer (Bruker, Germany) equipped with a standard bore (54 mm) 11.7 T magnet. 1 H-MRI and 31 P-MRI were recorded on the same spectrometer equipped with a Micro5 imaging probehead.
For MR spectroscopy, samples were transferred into a 5 mm NMR tube and filled with a mixture of 450 mL YPD media and 50 mL 2 H 2 O (CIL, USA) containing 100 mg methylene diphosphonic acid (MDP) (Fulka, Germany), as both a concentration and chemical shift standard. The assignments of the peaks in the 31 P-NMR spectrum were given by reference (17). The data were acquired by applying p/2 pulses at 1 s intervals with a sweep width of 16 181 Hz. A total of 16 384 complex points were acquired. For each measurement 512 transients were accumulated. Data acquisition and analysis were performed using XWinNMR software (Bruker). The quantities of inorganic polyP were estimated by the ratio of integrated peak areas of polyP and MDP.
For MRI, samples were transferred into a 0.8 mm outer diameter capillary glass tube (Hilgenberg, Germany). The bottom of the tube was sealed with a sealing compound (Fisher, USA). The tube was then centrifuged at 1000 r.p.m. for 3 min to pellet the yeast cells. The tubes containing test samples and a positional reference (water) were put into an 8 mm NMR tube, as depicted in Figure 3A. A 3D 1 H image was acquired to define the positions of samples. Subsequently, the 2D image of polyP in which the polyP signal was integrated longitudinally along the axis of each capillary tube was acquired by the chemical shift selective imaging method (CHESS) (18). For the CHESS, a p/2 pulse to excite ±1500 Hz region centered at the polyP signal with a Gaussian shaped and a non-selective rectangular p pulse were used. The sweep width was 5000 Hz, and TR and TE were 750 and 1.4 ms, respectively. The matrix size was 32 · 16 (zero-filled to 64 · 32 for analysis) with the field of view (FOV) of 1 cm · 1 cm. For each measurement, 256 transients were accumulated. No slice selective gradient was used. The obtained images were analyzed using ParaVision software (Bruker).

RESULTS AND DISCUSSION
Initial studies were carried out to determine whether VTC1 and VMA2, which encode subunits of the Vtc complex and the . The integral of the polyP peak was normalized to the MDP peak and the percentage of the maximum polyP signal (t ¼ 24 h after the shift from raffinose to galactose) was calculated. (C) Samples were prepared as described in panel (B) and analyzed by northern blot with a probe for VTC1. The VTC1 transcript was normalized to the ADH1 transcript and the percentage of the maximum value for each data was calculated. (D) Samples were prepared as described in panel (B) and used for immunoblot analysis with antibody to HA-tagged VTC1. VTC1 protein was quantified and normalized to TATA box binding protein (TBP). Values in bar graph are the percentage of VTC1 at t ¼ 0 (lane 1). Note that the strains expressing HA-tagged or untagged VTC1 protein produced almost the same amounts of polyP irrespective of endogenous or heterologous promoters. (E) Samples were prepared as described in panel (B) using yeast cells expressing HA-tagged VMA2 under the control of the GAL1 promoter. Northern blot analysis was carried out using probes for VMA2 (upper) or ADH1 (lower). VMA2 transcript was normalized to ADH1 and the percentage of maximum VMA2 expression (t ¼ 6 h after the shift from raffinose to galactose) was calculated. (F) PolyP was quantified by 31 P-MRS in yeast cells expressing HA-tagged VMA2 under the control of the GAL1 promoter. Samples were prepared at the indicated time after media switching as described in (B). The integral of the polyP peak was normalized to the MDP peak and the percentage of the maximum polyP signal (t ¼ 12 h after the shift from raffinose to galactose) was calculated.
V-ATPase, respectively, are suitable as quantitative reporter genes with MRS detection in intact cells. This was done by substituting six heterologous promoters of variable strength (19) for the endogenous promoter region of chromosomal VTC1 or VMA2 ( Figure 1A). PolyP was then quantified in yeast strains carrying the chromosomal reporter genes with heterologous or endogenous promoters. The results were evaluated to determine if the relationship between the expression of VTC1 and VMA2 and the amount of polyP was quantitative.
Yeast cells were cultured to mid-log phase in liquid YPD media and divided into two aliquots. One sample was analyzed by northern blot (Figure 1B and C) and the other was analyzed by 31 P-MRS ( Figure 1D and E). The northern blot shows very similar expression profiles for VTC1, VMA2 ( Figure 1B and C) and the mini-CLN2 reporter gene characterized previously (19), suggesting that no post-transcriptional regulatory mechanisms influence the steady-state level and/or stability of VTC1 or VMA2 mRNA. Furthermore, the amount of mRNA correlates well with the amount of polyP in the vacuole in all In contrast, the wild-type (WT) strains accumulate more polyP than expected based on the level VTC1/VMA2 mRNA ( Figure 3C). Although the mechanism for this enhanced accumulation is unknown, it is possible that the endogenous VTC1/VMA2 promoters are regulated by an uncharacterized feedback mechanism that leads to increased production of polyP.
In order to provide an accurate measure of gene expression in vivo, a reporter system must be temporally responsive; i.e. it must be able to respond quickly to rapid changes in gene expression. This aspect of the polyP system was tested using the inducible GAL1 promoter to drive gene expression. When the endogenous VTC1 or VMA2 promoters are replaced with the GAL1 promoter, gene expression can be rapidly turned on or off by changing the carbon source in the medium from raffinose to galactose or from galactose to glucose, respectively (20). Kinetic analysis (on-rate kinetics) showed that polyP begins to accumulate ( Figure 2A) by 1-2 h after cells are exposed to galactose and induction of VTC1 mRNA begins ( Figure 2C, left). In contrast, the level of polyP does not decrease for at least 24 h after mRNA synthesis is suppressed by changing carbon source ( Figure 2B, right); the concentration of polyP remains high, even though VTC1 mRNA returns to a low basal level within 1 h ( Figure 2C, right) and VTC1 protein is also suppressed within 2 h ( Figure 2D). One explanation for this result is that may be VTC1 plays a role in initiating vacuolar fusion (21) but does not play a direct role in synthesis of polyP; if this is true, then induction of VTC1 would stimulate accumulation of polyP in the vacuole, but subsequent suppression of VTC1 might not cause a decrease in the level of polyP.
Similar results were observed with VMA2, in that the amount of polyP correlated the amount of VMA2 transcript during induction but not during repression of VMA2 mRNA synthesis (data not shown). However, the correlation between gene expression and polyP improved when the reporter gene was modified with an N-terminal HA epitope tag. Thus, when an HA-tag was fused to the amino-terminus of VMA2, a decline in VMA2 mRNA correlated with a decline in polyP, with a lag of a few hours ( Figure 2E and F). This more rapid response might indicate that HA-tagged VMA2 has a shorter half-life than VMA2 (data not shown).
The VTC1 and/or VMA2 reporter system described above was then tested with MRI detection. The strains shown in Figure 1 were cultured in YPD to mid-log phase and transferred into 0.8 mm glass capillary tubes ( Figure 3A) and yeast cultures and a reference sample (i.e. water) were analyzed by MRI ( Figure 3B). Signals were integrated longitudinally in each capillary tube and are presented as a bottom view ( Figure 3B, right). The average intensity of the polyP image was calculated from 31 P-MRI data and the area corresponding to each sample was defined spatially using 1 H-MRI data ( Figure 3B, left).
Experimental values were measured by northern blot, MRS and MRI, and values were expressed as a percentage of the maximum value for each dataset. The results are summarized in Figure 3C and D. Figure 3D shows the correlation coefficients between northern blot and MRS or MRI methods. These results strongly suggest that gene expression can be quantified accurately by MRS and MRI using VTC1 or VMA2 as a reporter gene in yeast cells. To our knowledge, this is the first report describing a fully quantitative reporter system with MRI detection that can be used to monitor spatial and temporal variation in gene expression.
In higher eukaryotes, the endogenous level of polyP is much lower than in yeast (8). In addition, VTC1 orthologs have not been identified in mammals whereas mutations of VMA2 orthologs cause genetic disease in humans (22,23). Therefore, in order to apply a similar reporter system in mammalian cells, an exogenous MRI reporter gene may be required. Preliminary results suggest that the Escherichia coli PPK1 gene, which encodes polyP kinase, is a candidate reporter gene for use in mammalian cells. PPK1 converts the terminal phosphate of ATP to polyP (24), and expression of PPK1 from a mammalian promoter induced polyP at a level readily detected by MRS or MRI in mammalian cells (unpublished results). Unfortunately, it has not been possible to characterize a PPK1-based reporter system in yeast, because the endogenous background of polyP interferes with measurement of induced PPK1 activity (data not shown). Although organism-specific reporter genes may be needed for future studies in mammalian cells, the system described in this report should be widely applicable.
In summary, this study presents a novel MR-based method for quantifying temporal and spatial variation in gene expression. This method can potentially be used in any tissue of any organism. Furthermore, this method may have important applications in the context of high-throughput cell-based assays, which are used in drug discovery screening and in many other areas of basic and clinical research.