1 Laboratory of Animal Experiment for Disease Model, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan
2 G-in Techno Science, Sapporo 001-0015, Japan
3 Sankyo Labo Service Corporation, Tokyo 132-0023, Japan
Correspondence
Etsuro Ono
etsuro{at}imm.hokudai.ac.jp
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ABSTRACT |
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INTRODUCTION |
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During PRV latency, PRV DNA is retained in neurons of the trigeminal ganglion, and gene expression is restricted to a small region of the viral genome (Cheung, 1989a). RNA, termed latency-associated transcript (LAT), is synthesized in the opposite direction relative to IE and EP0 gene transcription (Fig. 1
A). Several sizes of LAT (8·4, 4·5 to 5·0, 2·0 and 0·95 kb) have been detected in latently infected porcine trigeminal ganglia (Cheung, 1989b
, 1991
; Priola & Stevens, 1991
). A spliced 8·4 kb poly(A) RNA, designated large latency transcript (LLT), has been detected in the trigeminal ganglia of both latently and lytically infected swine. Although several types of LAT are stable and accumulate in latent neurons, their function remains unclear. Several pieces of evidence suggest that LAT plays some role in facilitating reactivation of HSV from latency (Leib et al., 1989
; Hill et al., 1990
; Block et al., 1993
; Bloom et al., 1994
; Perng et al., 1994
; Krause et al., 1995
). On the other hand, some reports suggest that LAT is involved in establishment of latency (Sawtell & Thompson, 1992
; Garber et al., 1997
; Thompson & Sawtell, 1997
; Kramer et al., 1998
). Recently, it has been reported that LAT provides an anti-apoptotic function resulting in the survival of neurons during establishment of and/or reactivation from latency (Perng et al., 2000
; Inman et al., 2001
; Thompson & Sawtell, 2001
; Ahmed et al., 2002
).
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Transgenic technology has provided a means to examine the specific effect of promoter regulatory elements in vivo. In this study, we examined tissue-specific expression of the PRV LAP-CAT transgene in adult transgenic mice. Our results indicate that the PRV LAP directs neuron-specific expression in their trigeminal ganglia.
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METHODS |
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Detection of CAT expression in mouse tissues.
Mouse tissues were suspended into 30 vols of cold lysis buffer (137 mM NaCl, 10 % glycerol, 0·5 mM Na2VO4, 1 % NP40, 20 mM Tris/HCl, pH 7·5) containing 0·5 mM PMSF and homogenized with a high-speed homogenizer (Ultra-Turrax T25; IKA Labortechnik). Those homogenates were centrifuged at 12 000 g for 20 min, and the supernatants were collected and stored at -70 °C until analysis. Each tissue extract (approximately 150 µg in 200 µl) was used for CAT ELISA (Roche) to measure the levels of CAT in tissues of transgenic mice. CAT ELISA was performed as directed by the manufacturer. For normalization, total protein concentration in each tissue extract was determined with a Bio-Rad protein assay kit.
Western blot analysis.
Each tissue extract (400 µg) was used for immunoprecipitation with 40 µg of polyclonal sheep anti-CAT IgG (Roche) for 2 h at room temperature. Immunocomplexes were collected by centrifugation after incubating the extracts with 25 µl of Omnisorb (Calbiochem). The pellet was washed three times with PBS containing 0·05 % Tween 20 and suspended in 20 µl of 2x SDS sample buffer containing 5 % 2-mercaptoethanol. Samples were boiled at 100 °C for 5 min and separated by 16 % SDS-PAGE. Fifteen pg of recombinant CAT protein (Roche) was used for the positive control of the blot. The separated proteins were then transferred to Immobilon transfer membrane (Millipore). The membrane was treated with Buffer 1 (0·15 M NaCl, 0·1 M maleic acid, pH 7·5) containing 1 % Blocking reagent (Roche). After blocking for 30 min, the membrane was washed three times with Buffer 1 containing 0·03 % Tween 20 and incubated for 1 h with DIG-conjugated polyclonal sheep anti-CAT antibody (Roche) in Buffer 1 containing 1 % Blocking reagent. After incubation with the primary antibody, the membrane was washed as above and incubated with alkaline phosphatase-conjugated sheep anti-DIG antibody (Roche) for 30 min. The membrane was washed and equilibrated in Buffer 3 (100 mM NaCl, 100 mM Tris/HCl, pH 9·5). The antigen was detected using a CDP-star detection reagent (Amersham) as substrate.
Analysis of transgene expression by RT-PCR.
Total cellular RNA was isolated from various tissues of the transgenic mice using TRIzol reagent (Gibco BRL). For RT-PCR analysis, one microgram total RNA was digested with RNase-free DNase I (Gibco BRL) to remove any contaminating genomic DNA. The cDNA was synthesized from the DNase I-treated total RNA by MMLV reverse transcriptase (Gibco BRL) using oligo(dT) as a primer. The PCR reaction for the CAT gene was carried out as described above except that amplification was performed with 15 cycles instead of 30 cycles. Control samples without reverse transcriptase were amplified in parallel to confirm the absence of genomic DNA contamination. The PCR products were fractionated on 1·5 % agarose gel and analysed by Southern blot analysis. DIG-labelled DNA probes for detection of the transgene were derived from pCAT/Basic using the specific primers and a PCR DIG probe synthesis kit (Roche). Hybridization and detection of the transgene were performed as described previously (Ono et al., 1999).
In situ hybridization.
A HindIIIXbaI fragment containing the CAT gene was cloned into pGEM-4Z (Promega). Probes for sense and antisense CAT transcripts were prepared from the linearized plasmids using a DIG RNA labelling kit (Roche) according to the manufacturer's instructions. Mouse tissues were fixed with 4 % paraformaldehyde/PBS (PFA/PBS), and then embedded in paraffin. Sections (4 µm thickness) were collected onto glutaraldehyde-activated 3-aminopropyltriethoxysilane-coated slides and de-waxed in xylene before use. Tissue sections were treated with 0·2 M HCl and subsequently with 10 µg proteinase K ml-1 for 15 min at 37 °C. The sections were refixed in 4 % PFA/PBS for 10 min and treated with 0·25 % acetic anhydride in 0·1 M triethanolamine for 10 min. Riboprobes were diluted in hybridization buffer [50 % formamide, 500 mM NaCl, 10 mM Tris/HCl (pH 7·6), 1 mM EDTA (pH 8·0), 1x Denhardt's solution, 10 % (w/v) dextran sulfate, 200 µg yeast tRNA ml-1, 100 µg salmon sperm DNA ml-1], applied to the sections, and hybridized for 1618 h at 50 °C. After hybridization, the sections were washed twice with 0·2x SSC and blocked for 30 min at room temperature with 1·25 % Blocking reagent in Buffer 1. Bound probe was detected with alkaline phosphatase-conjugated anti-DIG antibody, as per the manufacturer's instructions (Roche).
Effect of reactivation stimuli on LAP activity.
Transgenic mice were sacrificed and their trigeminal ganglia were dissected out and explanted into the wells of a six-well plate. They were cultured for 24 h at 37 °C in Dulbecco's modified Eagle's medium (DMEM; Sigma) containing 10 % foetal calf serum and antibiotics. Transgenic mice were given an intravenous injection of 0·2 mg dexamethasone (Sigma) in water. Control transgenic mice were also given water without drug. Twenty-four hours after the dexamethasone injection, the trigeminal ganglia were removed. The levels of CAT in trigeminal ganglia of the transgenic mice were measured by using CAT ELISA described above.
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RESULTS |
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Expression of the CAT gene in transgenic mice
Expression levels of the CAT gene in various tissues of each transgenic mouse line were assessed by CAT ELISA. In three independent transgenic mouse lines (TgM16, TgM27 and TgM37), CAT expression was observed in neuronal tissues such as cerebral cortex, cerebellum, olfactory bulb, hippocampus and trigeminal ganglia (Table 1). Especially high CAT expression was observed in trigeminal ganglia. In contrast, CAT expression was hardly detected in non-neuronal tissues (Table 1
). A very small amount of CAT was detected in testis of TgM27 (Table 1
). Western blot analysis of tissue extracts of TgM16 with polyclonal antibodies to CAT identified a protein band (approximately 24 kDa) in the trigeminal ganglia extract (Fig. 2
A), which was consistent with the results of CAT ELISA analysis (Table 1
). No protein with the same size was detected in other tissue extracts, although low levels of CAT were detected in CAT ELISA analysis. To analyse expression levels of CAT mRNAs in various tissues of TgM16, an RT-PCR analysis was performed. The expected PCR product was detected in trigeminal ganglia (Fig. 2B
). In addition, weaker expression was observed in cerebellum. No PCR product was detected in other tissues. These results demonstrate that transgene expression was also strong in the trigeminal ganglia at the transcription level.
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DISCUSSION |
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It is unclear why TgM16, which carries the highest transgene copy number, has a more restricted pattern of transgene expression than the lower copy transgenic lines. Some variability in the level of expression between different lines of transgenic mice as was seen in these mice is common (Hammer et al., 1987; Koedood et al., 1995
; Mitchell, 1995
). The fact that all three of the lines share the key expression patterns which are the focus of this paper is taken as evidence that the expression pattern is the result of specific activation of the LAP by host cell proteins. It is possible that the chromatin structure or sequence elements within the chromosomal DNA adjacent to the transgene can influence this expression. The copy number of the inserted transgene is another variable which has been suggested as a possible cause of differing levels of expression in transgenic lines; however, in many cases, the expression level is unrelated to the copy number of the transgene (Hammer et al., 1987
; Koedood et al., 1995
; Mitchell, 1995
).
Many studies on the PRV LAT promoter using cultured cells have been performed with plasmids or recombinant viruses (Huang et al., 1994; Cheung & Smith, 1999
; Jin & Scherba, 1999
; Taharaguchi et al., 2002
). However, it is likely that transcriptional regulation of the LAT promoter in cultured cells is different from regulation in vivo. Transgenic experiments were intended to examine regulation of the LAP in the more relevant in vivo context. The PRV LAP has been shown to be significantly active in both neuronal and non-neuronal cells (Cheung & Smith, 1999
; Taharaguchi et al., 2002
), suggesting that the LAP may be a pan-specific promoter. However, we demonstrated in the present study that the LAP is a neuron-specific promoter. Our results indicate that the in vivo tissue-specificity of the LAP correlates well with the known target of PRV latent infection.
Several lines of evidence suggest that LAT plays some role in facilitating reactivation of HSV from latency (Leib et al., 1989; Hill et al., 1990
; Block et al., 1993
; Bloom et al., 1994
; Perng et al., 1994
; Krause et al., 1995
). Effects of putative reactivation stimuli on LAP activity were examined in the present study. However, explanation of the trigeminal ganglia and dexamethasone treatment did not affect the CAT expression levels. There are a number of possible explanations. The PRV LAP may not be the initial viral target for reactivation. It is possible that the trigeminal ganglion neurons which express the LAP-CAT transgene are different from the cells which maintain the latent viral genome. It is also possible that the latent viral genome DNA is regulated in a different way from the transgene which is within the host chromosome. The chromosomally located transgene may not be regulated precisely as the LAT promoter is in the context of the latent viral genome. Thus, the situation may indicate the limitations of the transgenic approach for studies on the regulatory mechanisms for LAT gene expression.
In the present study, it was demonstrated that expression levels of the transgene varied among neurons. These results may suggest that the level of activation of the PRV LAP can be altered by changes in the neuronal environment without any contribution from viral regulatory molecules. It is known that the transcription factors in a specific subset of neurons can be altered by changes in the neuron such as aging or differentiation (Herdegen & Leah, 1998). In fact, the HSV-1 ICP0 promoter in a specific subset of neurons was differentially regulated depending upon changes in the neuronal environment (Loiacono et al., 2002
).
The potential for neuronal regulation of PRV LAP in the absence of viral protein was shown in the present study. However, tissue-specific regulatory molecules which alter viral gene expression in neurons have not been identified. Further investigation of those regulatory molecules should be performed to elucidate fully PRV LAT gene expression. The specific expression of the PRV LAP-CAT transgene means that it is now possible to target expression of other heterologous genes to trigeminal ganglia.
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ACKNOWLEDGEMENTS |
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Received 3 January 2003;
accepted 2 April 2003.