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Article
A Neuron-Specific Host MicroRNA Targets
Herpes Simplex Virus-1 ICP0 Expression
and Promotes Latency
Dongli Pan,1 Omar Flores,2 Jennifer L. Umbach,2 Jean M. Pesola,1 Peris Bentley,1 Pamela C. Rosato,3 David A. Leib,3
Bryan R. Cullen,2 and Donald M. Coen1,*
1Department
of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA
3Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756, USA
*Correspondence: don_coen@hms.harvard.edu
http://dx.doi.org/10.1016/j.chom.2014.03.004
2Department
SUMMARY
INTRODUCTION
Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) cause a
variety of diseases ranging from common cold sores to rare
but fatal brain diseases such as encephalitis. HSV alternates
between two distinct infection programs, productive (lytic)
infection and latent infection. During lytic infection, infectious
virus is abundantly produced and many viral genes are highly
expressed. Immediate-early (IE) genes are expressed first and
activate the expression of subsequently expressed early (E)
and late (L) genes. Among the IE gene products is ICP0, which
acts as a promiscuous transactivator of genes introduced by
transfection or infection and is required for efficient lytic infection
in many cell types (Cai and Schaffer, 1992; Chen and Silverstein,
1992). ICP0 is a RING finger E3 ubiquitin ligase (Boutell et al.,
2002) that promotes degradation and dispersal of proteins that
446 Cell Host & Microbe 15, 446456, April 9, 2014 2014 Elsevier Inc.
the LAT locus encodes several miRNAs (Flores et al., 2013; Jurak
et al., 2010; Tang et al., 2008, 2009; Umbach et al., 2008), which
are differentially expressed during acute infection and latent
infection (Kramer et al., 2011; Tang et al., 2008; Umbach et al.,
2008) and have been hypothesized to play a role in repressing
lytic gene expression. Some host miRNAs have also been suggested to play a role in HSV infection (Hill et al., 2009; Mulik
et al., 2012; Zheng et al., 2011). However, repression of HSV
gene expression by host miRNAs has not been demonstrated.
Because HSV latent infection occurs in neurons, we wondered
whether a neuron-specific miRNA might repress lytic gene
expression. In a previous deep sequencing study of miRNAs
expressed during latency in mice (Umbach et al., 2008), we
noticed that reads of miR-138 were highly represented in trigeminal ganglia (TG). Earlier studies had found abundant expression
of miR-138 in brain or neuroblastoma cell lines relative to other
tissues or cell lines (Landgraf et al., 2007; Obernosterer et al.,
2006). This miRNA was later found to regulate dendritic spine
morphogenesis in the mouse brain by modulating APT1 (Banerjee et al., 2009; Siegel et al., 2009), and its expression in mouse
sensory ganglia was reduced when the gene encoding the
miRNA-processing enzyme Dicer was disabled in a subset of
sensory neurons (Zhao et al., 2010).
The neuronal expression of miR-138 prompted us to search
HSV-1 and HSV-2 genomes for complementary sequences,
and we identified ICP0 mRNA as a potential target. Notably,
ICP0 is required for efficient acute ganglionic replication and
reactivation from latency (Cai et al., 1993; Halford and Schaffer,
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onic titers (Figures 4A and 4B). Eye swab titers decreased from 1
to 3 dpi but increased from 3 to 5 dpi, which might suggest reinfection of the cornea from the TG, before decreasing again to
very low levels at 7 dpi. TG titers decreased from 3 dpi to very
low levels at 7 dpi (Figure 4B), suggesting that at 7 dpi acute
ganglionic replication is waning and latency is being established.
Eye swab and ganglionic titers were also similar between M138and R138-infected mice (data not shown).
Next we analyzed TG samples obtained at 7 dpi from mice
infected with WT, M138, or R138 for viral DNA levels by qPCR,
and RNA levels by qRT-PCR. The M138 mutations had little or
no effect on viral genome levels or levels of the stable LAT intron
(Figure 4C). However, they significantly increased the levels of
ICP0 transcripts (Figure 4C). Considering ICP0s role in activating lytic genes, we also analyzed the levels of other lytic transcripts, namely ICP4 (IE), ICP27 (IE), tk (E), and gC (L). The M138
mutations increased levels of all four of these lytic transcripts by
about 3-fold (2.3- to 3.8-fold; Figure 4C), and all of these differences were statistically significant (p < 0.05), except the difference between the ICP4 levels of WT and M138 (p = 0.06). There
were no significant differences in transcript levels in ganglia
infected with WT versus R138 viruses (Figure 4C), indicating
that the increases in transcript levels in M138-infected mice
were due to the engineered mutations. We performed an independent replicate of this experiment, which yielded very similar
results (Figure S3). We also found that ICP0 transcript levels
were significantly correlated with levels of each of the other lytic
transcripts assayed, but not with LATs, consistent with previous
studies suggesting divergent pathways for acute and latent infections (Margolis et al., 1992; Speck and Simmons, 1991).
Notably, the correlation between ICP0 and the other lytic transcripts was increased by the M138 mutations (Figure 4D; Table
S1). Taken together, the results show that the miR-138 target
site mutations in ICP0 mRNA increase lytic gene expression
during establishment of latency in mouse TG.
The M138 Mutations Increase Morbidity and Mortality
Infection of CD-1 mice by HSV-1 strain KOS usually results in low
mortality and thus efficient establishment of latency (Leib et al.,
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Figure 4. Virus Replication and Gene Expression during Acute Infection of Mice
(A) Virus replication in the eye. Each point represents the geometric mean titer of at least 15 eye swabs.
(B) Virus replication in the TG. Each point represents the geometric mean titer of at least 10 TG. There are no statistically significant differences between the titers
of WT and M138 viruses in (A) and (B).
(C) Viral genome and transcript levels in TG at 7 dpi. Each point represents a value from one TG. Virus names are shown at the bottom, and the molecules assayed
are labeled at the top of each graph. Vertical axes show logarithmic values, and RNA levels were normalized to viral genome levels. The horizontal lines represent
the geometric means (any undetectable values were included in the calculations of means as logs of the detection limits). p values for the two viruses compared
are shown on top of the brackets.
(D) Correlations between transcript levels in TG infected by the viruses indicated at the top of each plot at 7 dpi. The levels of gC transcripts or LATs were plotted
against the level of ICP0 transcripts in the same TG. One TG that exhibited undetectable levels of gC and ICP0 transcripts was excluded from the plots. R2 and p
values for each correlation are shown at the upper left corners of the plots. See also Figure S3 and Table S1.
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Figure 5. The M138 Mutant Virus Causes Increased Mortality and Disease
(A) Survival curves of mice infected by the viruses indicated in the key. Percentages of live mice as a function of time were plotted. The numbers of mice studied
and the p values for the differences between viruses are shown below the curves.
(B) Mice showing encephalitis symptoms. The bars represent percentages of mice at 17 or 25 dpi that showed impaired mobility, i.e., partial paralysis or lethargy.
The viruses and the numbers of mice studied for each virus are indicated to the right of the graph. p values for statistical comparisons are shown above the
brackets, and those with statistical significance are labeled with asterisks below the brackets. Since two Fishers exact tests were performed for each time point,
the threshold alpha level below which a p value is considered significant is adjusted from 0.05 to 0.0253 in order to control for multiple comparisons.
(C) Titers of WT and M138 viruses in mouse brain at 7 dpi. Each point represents a value from one brain. The horizontal lines represent the geometric means of
titers. See also Figure S4.
on average for all four genes analyzed than did the WT and
R138 viruses (data not shown). However, these differences
were not statistically significant. Nevertheless, all four lytic transcripts were detectable in a higher fraction of M138-infected TG
than in WT- or R138-infected TG (Figure 6A). A separate experiment performed with fewer mice but using identical conditions
also resulted in a higher fraction of M138-infected TG having
detectable ICP0 and ICP27 transcripts than WT- and R138infected TG (data not shown). Pooling the data from the two
experiments, M138 infection was associated with increased
fractions of TG exhibiting detectable transcripts for all four lytic
genes, with the increases being statistically significant for
ICP27 (Figure 6B). Analyzing only those TG in which both transcripts could be detected, ICP0 levels were uncorrelated with
LAT levels, but were significantly correlated with the levels of
the other three lytic transcripts in M138-infected TG (Figure 6C).
These data suggest that the miR-138 target site mutations may
also increase expression of lytic genes during maintenance of
latency.
DISCUSSION
The mechanisms that promote HSV-1 latency have remained
largely obscure, although roles for neuronal factors and for
repression of ICP0, which is important for antagonizing host
silencing mechanisms and for efficient replication and reactivation in ganglia, have long been suspected (reviewed in Roizman
et al., 2013; Boutell and Everett, 2013). Here we found that a
neuron-specific host miRNA, miR-138, binds to two target sites
in ICP0 mRNA and can repress ICP0 expression in both transfected and HSV-1-infected cells. This repression is dependent
on the miR-138 seed sequence and its target sites in ICP0
mRNA. Importantly, mutations of these miR-138 target sites do
not affect ICP0 expression in cells in which miR-138 is not abundant. However, in cells into which exogenous miR-138 is introduced, and in Neuro-2A cells and in mouse TG tissue where
endogenous miR-138 is abundant, these mutations result in
increased expression of ICP0 and other lytic genes. By far the
simplest interpretation of these results is that high levels of
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Figure 6. Viral Genome Levels and Gene Expression during Latent Infection of Mice
(A) Viral genome and transcript levels at 32 dpi. Values are presented as in Figure 4C, except that mean values are not indicated in the plots for viral lytic
transcripts, because the large numbers of undetectable values make difficult meaningful calculations of means.
(B) Percentages of TG exhibiting detectable lytic transcripts. Each vertical bar represents the value for each virus (indicated in the key) and each RNA (below the
graph). Since two Fishers exact tests were performed for each gene, the threshold alpha level below which a p value is considered significant is adjusted from
0.05 to 0.0253 in order to control for multiple comparisons. Therefore, only p values below 0.0253 have been displayed.
(C) Correlation between transcript levels in M138 virus infected TG at 32 dpi. The plots are presented as in Figure 4D. Only TG exhibiting detectable transcripts for
both RNAs being compared are included in the analysis.
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an Illumina TrueSeq small RNA kit was used for cDNA preparation and an
Illumina HiSeq 2000 was used for sequencing. Resulting reads were then processed using the FASTX-toolkit and aligned to the HSV-1 genome using Bowtie (Langmead et al., 2009). Reads of at least 15 nt in length, with minimum 50
reads total, were retained if they mapped to HSV-1 with %2 mismatches.
Transfection
For cotransfection using a miR-138 mimic, 293T cells in each well of a 24-well
plate were cotransfected with 75 ng of ICP0 (WT) plasmid and 4 pmol of
miScript miR-138 or miR-M138 mimic (QIAGEN). Cells were harvested at
24 hpi. For cotransfection using a miR-138 expression plasmid, 293T cells in
each well of a 6-well plate were cotransfected with 0.5 mg of ICP0 (WT or
M138) plasmid and 2.5 mg of pcDNA-miR138, with an additional 1 mg of GFP
expression plasmid, which served as a transfection and loading control. Cells
were harvested at 48 hr posttransfection. For transfection-infection, 293T cells
in each well of a 24-well plate were transfected with 8 pmol of the miR-138 or
miR-M138 mimic with or without 16 pmol of miScript miR-138 inhibitor or a
control scrambled inhibitor (QIAGEN). After 8 hr, the cells were infected with
virus at an moi of 0.1 and lysed for analysis at 48 hpi. All transfection procedures used Lipofectamine 2000 (Invitrogen) according to the manufacturers
protocol.
Western Blotting
Western blots were performed as previously described (Pan and Coen,
2012b). The following antibodies and dilutions were used for probing the blots:
ICP0 mouse monoclonal antibody (Virusys), 1:8000; GFP monoclonal (Santa
Cruz), 1:20,000; ICP4 mouse monoclonal (Abcam), 1:1000; ICP27 mouse
monoclonal (Virusys), 1:1000; TK mouse monoclonal (a generous gift from
Bill Summers, Yale University), 1:1000; gC mouse monoclonal (Fitzgerald),
1:1000; actin antibody (Sigma), 1:10,000; and HRP-conjugated goat antimouse antiserum (SouthernBiotech), 1:2000. Images were visualized by either
using a Syngene G:Box gel imaging system or film.
Animal Procedures
Animal housing and experimental procedures were approved by the Institutional Animal Care and Use Committee of Harvard Medical School in accordance with federal guidelines. Male CD-1 mice (Charles River Laboratories)
were anesthetized by intraperitoneal injection of 100 mg/kg ketamine (Fort
Dodge Animal Health) and 10 mg/kg xylazine (Lloyd Laboratories). 2 3 105
pfu of virus in 3 ml was dropped onto each scarified cornea of 7-week-old
mice. For eye swab collection, mice were anesthetized transiently in an induction chamber with isoflurane (3% in oxygen 0.5 ml/min) using an anesthesia
machine (Colonial Medical Supply). Both eyes of each mouse were swabbed
with cotton-tipped applicators, which were suspended in 1 ml of cell culture
media. For TG or brain acquisition, mice were sacrificed by cervical dislocation
while under inhalation anesthesia with isoflurane as above, and the TG or brain
was rapidly removed and placed on dry ice before storing at #80" C. TG and
brain were homogenized in cell culture media for titers. Mice were observed
daily, and mortality figures include mice that had died and one that was moribund and therefore euthanized. At 17 and 25 dpi each mouse was scored for
its mobility (partial paralysis or lethargy). All of these mice were recovering by
32 dpi.
Statistical Analysis
Statistical analysis was performed using Prism 6 (GraphPad). Titers and DNA
and RNA levels were analyzed by Kruskal-Wallis tests for all virus genotypes,
along with Dunns post tests for M138 versus WT and M138 versus R138
(controlling for multiple comparisons). Survival curves were analyzed by the
log rank (Mantel-Cox) test. The number of mice with encephalitis sequelae
at 17 and 25 dpi or of TG with detectable transcripts at 32 dpi was analyzed
by Fishers exact test. The covariance of transcript levels was evaluated using
Pearson correlations.
SUPPLEMENTAL INFORMATION
Supplemental Information includes four figures, one table, and Supplemental
Experimental Procedures and can be found with this article at http://dx.doi.
org/10.1016/j.chom.2014.03.004.
ACKNOWLEDGMENTS
We thank Seamus McCarron for technical assistance, Angus Wilson and
Michael OBrien for kindly providing total RNA from rat SCG neurons, Bill
Summers for TK antibody, Jeremy Kamil and Igor Jurak for advice on the mutagenesis strategy, Martha Kramer for plasmids for transcribing ICP27 and gC
mRNA standards, John Carbone for designing the ICP27 primers for qRTPCR, David Knipe for helpful discussions, the National Institutes of Health
for funding (P01 NS035138, R21 AI105896, and P01 AI098681 [D.M.C.]; P01
AI098681 [D.A.L.]; R01 AI0973376 [B.R.C.]), NCI training grant T32
CA009111 (O.F.), and the Geisel School of Medicine Microbiology and Molecular Pathogenesis Program training grant T32 AI007519 (P.C.R.).
Received: October 15, 2013
Revised: January 15, 2014
Accepted: February 18, 2014
Published: April 9, 2014
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Supplemental Information
A Neuron-Specific Host MicroRNA Targets
Herpes Simplex Virus-1 ICP0 Expression
and Promotes Latency
Dongli Pan, Omar Flores, Jennifer L. Umbach, Jean M. Pesola, Peris Bentley,
Pamela C. Rosato, David A. Leib, Bryan R. Cullen, and Donald M. Coen
Supplemental Figures
Figure S1, related to Figure 2
results in replacement of copy 2 with the modified HSV-1 sequences containing the M138
mutations, resulting in Kans and Zeor colonies identified using replica plating. Step 4, the Zeo
cassette in copy 1 is replaced with the Kan cassette used in step 2. Step 5, the Kan cassette in
copy 1 is replaced with the modified HSV-1 sequences containing the M138 mutations, as in step
3. The same procedures were used for generation of the R138 BAC from the M138 BAC except
that WT sequences were used to replace sequences containing the M138 mutations. (B) Analysis
of BACs from each step of the generation of the M138 mutant. In all 3 gels, the left-most lane is
a DNA ladder with sizes indicated in kilobasepairs (kb), and BAC names are labeled on top of
the gels. Top gel, PCR products from each BAC using primers spanning the mutation site
(insertion of Kan or Zeo or replacement with M138 sequences) were analyzed using a 1 %
agarose gel, and stained with ethidium bromide. The arrows indicate positions of bands
corresponding to no insertion (Original) or insertion of Kan or Zeo. Bottom left gel, The
indicated BAC genomes were digested with EcoRI and analyzed using a 0.75% agarose gel,
stained with ethidium bromide. Insertion of a Zeo cassette (0.4 kb and containing an EcoRI site
in copy 1 converted an 18.2 kb band to a 11.1 kb and a 7.5 kb band in M138Z, M138ZK and
M138ZM as indicated by arrows. Insertion of a Kan cassette (about 1 kb) in copy 1 converted the
18.2 kb band to one with lower mobility in M138KM. Bottom right gel, The indicated BAC
genomes were digested with HindIII, and analyzed using a 0.75% agarose gel stained with
ethidium bromide. Insertion of a Kan cassette (~1 kb, containing a HindIII site) in copy 2
resulted in the conversion of a 34.6 kb band in M138Z to a 22.6 kb and a 13.0 kb band in
M138ZK as indicated by arrows pointing from the left. However, since the original 34.6 kb band
overlaps with another band of a similar size (only ~0.6 kb different), its disappearance is not
readily discernible. Insertion of a Kan cassette in copy 1 resulted in conversion of a 26.6 kb band
in M138ZM to a 20.1 kb and a 7.5 kb band in M138KM as indicated by arrows pointing from the
right. The same patterns of PCR and digestion products were detected during the generation of
the R138 BAC from the M138 BAC (not shown). (C) M138 virus exhibits no defect for
replication in cell culture. In each plot of viral replication kinetics, the cells and MOIs used are
indicated at the top. Each point represents the geometric mean titer measured in triplicate. (D)
Magnitudes of effects on viral gene expression. The experiment in Figure 3C was repeated for
the 7 and 12 h time points, and analyzed using SDS-PAGE and Western blotting using antibodies
against the proteins indicated to the left alongside a dilution series prepared from cells infected
with M138. Two fold-dilution is indicated as M138/2, etc. (E) Transfected miR-138 reduces
ICP0 expression in HSV-1 infected 293T cells. 293T cells were transfected with mimics of miR138 or miR-M138 8 hours prior to infection with WT or M138 viruses as indicated at the top of
the figure. At the times indicated after infection, cells were lysed and ICP0 levels (upper panels)
and actin levels as loading controls (lower panel) were analyzed by Western blot.
Figure S3. Replicate experiment measuring viral genome and transcript levels 7 days after
infection of mice with WT, M138, or R138. The experiment was performed and the data are
presented in exactly the same way as the experiment in Figure 4C.
Figure S4. Effects on mortality following different routes of inoculation. (A) Mortality of
infected mice following corneal inoculation in the experiment shown in Fig. 5A. Vertical bars
represent percentages of mice that died before 32 dpi. The viruses are indicated at the bottom.
(B). Survival curves for mice infected intracranially by the viruses indicated. Total numbers of
mice used and the P value by Log-rank test are shown for each virus. The survival curve (plotted
according to criteria described in Supplementary Experimental Procedures) represents combined
data from two independent experiments.
Supplemental Tables
Table S1, related to Figure 4. R2 values for correlation between ICP0 transcripts and other
transcripts in different TG at 7 dpi.
Transcript WT
M138 R138
ICP4
0.54 0.81
0.61
ICP27
0.34 0.68
0.63
tk
0.30 0.58
0.52
gC
0.47 0.73
0.41
LATs
0.00 0.09
0.13
using SYBR green PCR master mix (Applied Biosystems) on an Applied Biosystems
StepOnePlus real-time PCR system following the manufacturers instructions. Optimized PCR
reaction conditions including primer sequences are provided below. Standard curves were linear
in all cases (with R2 values over 0.99) and the values reported for samples were all within the
linear ranges, except for those below the detection limits , (i.e., the lowest points of the linear
ranges of standard curves). The ICP0, ICP27, tk and gC assays detection limits were 100
copies/TG, and ICP4 assay's was 1000 copies/TG.
Luciferase assays
Reporter vectors were generated using the renilla luciferase (RLuc) vector pNL-SIN-CMVRLuc, as described (Gottwein et al., 2006). Briefly, two tandem fully complementary synthetic
binding sites for miR-138 were inserted using ClaI and XbaI sites present in the 3UTR of RLuc.
ICP0 3UTR RLuc was generated by PCR amplification of infected cell genomic DNA to yield a
200-bp fragment derived from the HSV-1 ICP0 3UTR, extending from 120,463 to 120,673
bases (KOS GenBank: JQ673480.1) in the viral genome, which fully encompasses both
predicted miR-138 target sites as well as flanking sequences. This DNA fragment was cloned
into the ClaI and XbaI sites present in pNL-SIN-CMV-RLuc. RLuc expression constructs (50
ng) were co-transfected with 50 ng of the internal control firefly (FLuc) expression vector pGL3
(Promega) into a 24-well plate seeded with miR-138- transduced 293 cells, and RLuc and FLuc
luciferase activities determined at 24 h post-transfection using a dual luciferase assay kit
(Promega).
Intracranial inoculation.
Male CD-1 mice (Charles River Laboratories) were infected at 4.5 weeks and housed in the
Center for Comparative Medicine and Research at The Geisel School of Medicine at Dartmouth.
Mice were anesthetized with 4% isoflurane and injected intracranially with 5000 PFU of HSV-1
M138 or R138 in a volume of 10 l Phosphate Buffered Saline (PBS) using a Hamilton syringe
with a 26-gauge needle. Mice were weighed daily and mortality figures were generated from
mice that were euthanized after 25% loss of body weight from day 0 of the experiment. Mice
were housed, treated, and euthanized in accordance with all Federal and University policies.
Forward primer
Reverse primer
GTCAGGATCCGGGATGGGGAA
GTCAGAATTCTGGAGCATTT
GTTCACAAAG
GTGTTGGGGG
TCTGGCCGCGCCCACTACAGC
ATTGTCAGCTGTAGTGGGCG
TGACAAT
CGGCCAGA
AAGACACGGGCACCACACAG
CCCTCAGCTGTGTGGTGCCCG
CTGAGGG
TGTCTT
Step
Forward primer*
Reverse primer
From
Amplify and
GGGGGTCGGGCGCTGGGTGGTCTCTGGCC
AATCCCCTGAGTTTTTTTTTATTAGGGCCA
WT to
incorporate
GCGCCCACTACAGCTGACAATCCGTGTCG
ACACAAAAGACCCTCAGCTGTGTGGTGC
M138
Zeo cassette
GGGAGGTGGAATTCtgttgacaattaatcatcggcat
CCGTGTCTTTCACTTTtcagtcctgctcctcggcca
Amplify and
GGGGTCGGGCGCTGGGTGGTCTCTGGCCG
TCCCCTGAGTTTTTTTTTATTAGGGCCAAC
incorporate
CGCCCACTACAGCTGACAATCCGTGTCGG
ACAAAAGACCCTCAGCTGTGTGGTGCCC
Kan cassette
GGAGGTGGAAAGTGAAAGACACGGGCAC
GTGTCTTTCACTTTCCACCTCCCCGACAC
CACACAGCTGAtagggataacagggtaatcgattt
GGATTGTCAGCgccagtgttacaaccaattaacc
From
Amplify and
GGGGGTCGGGCGCTGGGTGGTCTCTGGCC
AATCCCCTGAGTTTTTTTTTATTAGGGCCA
M138 to
incorporate
GCGCCCACTACACCAGCCAATCCGTGTCG
ACACAAAAGACCCGCTGGTGTGTGGTGC
R138
Zeo cassette
GGGAGGTGGAATTCtgttgacaattaatcatcggcat
CCGTGTCTTTCACTTTtcagtcctgctcctcggcca
Amplify and
GGGGTCGGGCGCTGGGTGGTCTCTGGCCG
TCCCCTGAGTTTTTTTTTATTAGGGCCAAC
incorporate
CGCCCACTACACCAGCCAATCCGTGTCGG
ACAAAAGACCCGCTGGTGTGTGGTGCCC
Kan cassette
GGAGGTGGAAAGTGAAAGACACGGGCAC
GTGTCTTTCACTTTCCACCTCCCCGACAC
CACACACCAGCtagggataacagggtaatcgattt
GGATTGGCTGGgccagtgttacaaccaattaacc
*In lower case are the sequences overlapping Zeo or Kan gene. In italics are the EcoRI
restriction sites. In bold letters are the mutated bases.
Forward primer
Reverse primer
Forward
Reverse
Annealing
(nM)
(nM)
temperature
(C)
mRNA
ICP0
AGCGAGTACCCGCCGGCCTG
CAGGTCTCGGTCGCAGGGAAAC
200
100
72
ICP4
TCGAGAGTCCGTAGGTGAC
TTGTTCTCCGACGCCATC
500
500
65
ICP27
GTGTGCAGCCGTGTTCCAA
AGCGACCGGGCCCGAATC
300
300
60
Tk
ACCCGCTTAACAGCGTCAACA
CCAAAGAGGTGCGGGAGTTT
100
200
58
gC
GCCCATTTCGTACGACTACA
GGTGCTCTAGAACGGGAATC
300
300
60
Mouse
GAAGGTCGGTGTGAACGGATT
GCCTTGACTGTGCCGTTGAA
300
300
60
GAAGGTCGGAGTCAACGGATT
GCCTTGACGGTGCCATGGAA
LAT
CAGACAGCAAAAATCCCCTGAGT
GGGACGAGGGAAAACAATAAGGG
300
300
60
Viral
Same as tk
Same as tk
300
300
60
TCCGGCAGCCCTCTAGT
TAGGATGACACTCGGGTATAGAC
300
300
60
GAPDH
Human
GAPDH
DNA
genome
(tk)
Cellular
DNA
(adipsin)
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