antigens in serum may only indicate exposure to the parasite or a parasite with similar antigens and not necessarily indicate an active infection ( Vardeleon et al., 2001). Because of this, we believe that 1:50 is the cutoff more suitable for screening herds when seeking for animals that are

challenged by Neospora sp. The high frequency of antibodies against Neospora sp. found in this study highlights the relevance of such protozoan, as well as the need for control programs, since seropositive herds of horses may have reproductive problems as consequence ( Pitel et al., 2003), or EPM ( Finno et al., 2007 and Finno et al., 2010). Mare samples that reacted positively in IFAT were titrated to assess the titer influence in transplacental transmission, but no difference was observed in Temozolomide order the vertical transmission rate as the titer. In samples from newborns, we can observe 51 animals reacting to Neospora sp. antigens before ingesting colostrum. This fact shows that the intrauterine infection occurred in 25.1% of pregnancies ( Table 1) since there is no transfer of immunoglobulins through the placenta of the mare due to its diffuse microcotyledonary epitheliochorial conformation ( Abd-Elnaeim et al., 2006), besides the fact that the equine fetus is capable of forming a humoral immune response if exposed to an antigen after 180 days of gestation ( Cook et al., 2001).

Although the prevalence in mares is higher than in foals (χ2 = 17.9, P < 0.0001) ( Table 1), suggesting that most infections are postnatal, the

results allow us to affirm that the vertical infection may be Ruxolitinib supplier an important form of Neospora sp. transmission in horses, as discussed by Locatelli-Dittrich et al. (2006), Toscan et al. (2010), and verified by Pusterla et al. (2011) in three pre-colostral seropositive foals. As expected, positive mares are more likely to transmit neosporosis to their offspring by transplacental route than seronegative ones (OR = 6.07, CI = 95%, from 2.44 to 15.08) (Table 1) due to the fact that seropositives mares are carriers of Neospora sp. However, six foals born from seronegative mares had detectable levels of anti-Neospora sp. IgG (≥1:16) before the ingestion of colostrum, this fact can for be attributed to antibody titer fluctuations of the mother during pregnancy ( Kormann et al., 2008). These six mares probably were infected by Nesopora sp., but at blood collection, their antibody levels are not detectable by IFAT. Even though endogenous challenge is proven to occur in mares, we must consider that mares were tested only at parturition without registers about reproductive failure due to neosporosis during pregnancy. Moreover, cross-reaction could occur in N. hughesi infected horses when tested using N. caninum antigens, as used in this study ( Gondim et al., 2009).

It has been emphasized (Behrens et al., 2007 and Yu and Dayan, 2005) that uncertainty may be used to the advantage of learners, allowing them to optimally weigh new data against old when updating their beliefs. One approach, which could be regarded as a form of novelty detection, suggests that learners quantify at each time point the likelihood that the statistics underlying the environment have changed based on the current sample (Nassar

et al., 2010, Payzan-LeNestour PD 332991 and Bossaerts, 2011 and Yu and Dayan, 2005). This quantity, termed unexpected uncertainty, can be used to flexibly modulate the weight given to new data as evidence for such a change varies. The computation of unexpected uncertainty is nontrivial, because improbable data samples may be attributed to a change in the statistics underlying the environment, or alternatively to the known unreliability of predictive relationships, dubbed expected uncertainty (Yu and Dayan, 2005). Importantly, the

definition of unexpected uncertainty does not imply that the agent is unaware that his environment is subject to change. Instead, selleck products a data sample with high unexpected uncertainty indicates that it is surprising given the cue-outcome association acquired through sampling, even when expected uncertainty, or the known, learned unreliability of this association, is accounted for. One form of expected uncertainty is risk, or the inherent stochasticity

of the environment that remains even when Phosphatidylinositol diacylglycerol-lyase the contingencies are fully known. For example, when sampling from an environment in which reward is delivered 50% of the time versus one in which reward is delivered 95% of the time, risk is higher in the former case. The perceptions of risk and unexpected uncertainty are antagonistic (Yu and Dayan, 2005) in the sense that when risk is high, as in the former case, changes in the environment are hard to detect and hence, unexpected uncertainty is low, whereas when risk remains low, as in the latter example, changes in the environment lead to strong increases in unexpected uncertainty. Unexpected uncertainty is also influenced by estimation uncertainty or the imprecision of the learner’s current beliefs about the environment (Chumbley et al., 2012, Frank et al., 2009, Payzan-LeNestour and Bossaerts, 2011, Prévost et al., 2011 and Yoshida and Ishii, 2006), which is also referred to as second-order uncertainty (Bach et al., 2011). If beliefs are acquired through learning as opposed to instruction, this quantity decreases with sampling. When estimation uncertainty is high, improbable samples may be partially attributed to the agent’s inaccurate beliefs about the structure of the environment, rather than to a change in that structure. Recent behavioral work suggests that subjects’ choices may indeed reflect a learning scheme that makes use of unexpected uncertainty (Nassar et al.

One obvious question, based upon our Venetoclax in vitro results is: Why does the cell need such a complicated pathway for adjusting ataxin-7 expression? Ataxin-7 is a core, and likely essential, component of different ubiquitously expressed transcription coactivator complexes (Helmlinger et al., 2004, Palhan et al., 2005 and Zhao et al., 2008). As deletion of the ataxin-7 ortholog Sgf73 eliminates Ubp8-mediated histone deubiquitination in yeast (Köhler et al., 2008), and knockdown of ataxin-7 results in disassembly of the STAGA complex in mammals (Palhan et al.,

2005), tight regulation of ataxin-7 expression could be a mechanism for controlling the activity of these coactivators. CTCF is a master regulator of transcription, and its expression Pictilisib ic50 cannot be significantly adjusted without killing the cell. However, minor changes in CTCF levels, or binding activity, could have a dramatic impact upon the transcriptional activity of the

cell through its regulation of ataxin-7 expression, since ataxin-7 expression alterations would be amplified by affecting the stability and function of entire co-activator complexes. Thus, CTCF control of ataxin-7 levels could serve as a rheostat for setting global transcription activity status for the cell. Another important implication of our work is its relevance to SCA7 disease pathogenesis and repeat disease biology. As we have shown, expansion of the ataxin-7 CAG repeat tract reduces SCAANT1 promoter activity, resulting in minimally detectable levels of SCAANT1 from the expanded allele in SCA7 patient fibroblasts. This reduction in SCAANT1 expression derepresses the ataxin-7 alternative promoter, and significantly boosts the level

of ataxin-7, creating a feed forward effect that agonizes the SCA7 disease pathway by favoring increased production of mutant ataxin-7 protein (Figure 8). Although we cannot exclude a role for altered transcript stability in this process, a survey of histone posttranslational modifications in our SCA7 transgenic mice revealed repressive chromatin modifications in the alternative promoter when Oxymatrine SCAANT1 transcription is robust, indicating that transcriptional activity is likely more important than transcript stability in controlling ataxin-7 sense expression. As bidirectional transcription in association with CTCF binding is emerging as a common feature of repeat disease loci, our findings could be applicable at other repeat disease loci, including loci that have not been carefully screened for antisense transcripts. Furthermore, the existence of regulatory bidirectional transcription may offer an entry point for therapeutically modulating ataxin-7 expression at the RNA level.

, 2001), and suggested for VAMP7 (Pryor et al., 2008). In our experiments expression of truncated vti1a OSI 906 triggered a prominent augmentation of baseline levels of spontaneous release

detected electrophysiologically, suggesting the existence of a mechanism that may circumvent potential autoinhibition of vti1a, akin to earlier proposals of VAMP7 as well as syntaxin1 function (Dulubova et al., 1999 and Pryor et al., 2008). Indeed, VAMP7-pHluorin lacking the longin domain has an increased rate of spontaneous exocytosis compared to full-length VAMP7 (Hua et al., 2011). In earlier studies, individual KOs of vti1a and vti1b did not reveal significant phenotypes, whereas the double KO of these genes triggered severe abnormalities in neuronal development (Atlashkin et al., 2003 and Kunwar et al., 2011). Well-characterized roles of these proteins in constitutive endosomal trafficking may complicate the evaluation of their loss-of-function phenotypes with respect

to their specific role in synaptic transmission. Nevertheless, the shRNA-based loss-of-function experiments showed a specific reduction in spontaneous release, indicating that shRNA-based KD of vti1a can provide insight into synaptic role(s) of vti1a without compromising neuronal survival. Typically, incomplete reductions in SNARE proteins do not result in discernable phenotypes as these proteins are present in excess quantities beyond minimum requirements (Bethani et al., 2009); therefore, it is noteworthy that in our hands KD of click here vti1a gave rise to a distinct synaptic phenotype. This finding suggests that the amount of vti1a present on vesicles may encode a rate-limiting step regulating levels of spontaneous release. At the level of the whole organism, to a selective deficit in spontaneous neurotransmitter release may not give rise to an overt phenotype.

For instance, mice that lack double C2 domain 2b (doc2b) show a specific deficit in Ca2+-dependent regulation of spontaneous release without overt alterations in behavior (Groffen et al., 2010 and Pang et al., 2011). However, spontaneous release deficits in doc2b KOs and those potentially associated with vti1a or vti1b single KOs may lead to subtle changes in behavior that would require closer examination. Indeed, spontaneous neurotransmission has recently been shown to mediate the fast-acting antidepressant action of NMDA receptor blockers on mouse behavior (Autry et al., 2011). A growing number of studies suggest that spontaneous neurotransmitter release can be regulated independently of evoked neurotransmission (Ramirez and Kavalali, 2011). Identification of a distinct pool marked by vti1a should be taken as one factor contributing to a larger context of other observations, which together can explain why spontaneous and evoked SV trafficking processes are functionally segregated.

, 2006; Thompson and Best, 1989; Crizotinib order Ylinen et al., 1995), but intermittent periods of reduced CA1 ensemble firing rates of 1 Hz and below may also occur during other states of network activity (Thompson and Best, 1989). On the other hand, during theta rhythmic ensemble activity that occurs during explorative behavior and rapid-eye-movement sleep (O’Keefe and Nadel, 1978), CA1 pyramidal neurons repetitively discharge single or bursts of action potentials at frequencies of 4–12 Hz (Csicsvari et al., 1999; Ranck, 1973). Under these conditions, our data predict a strong attenuation of

proximal inhibition already after the first few theta cycles, and a concomitantly higher contribution of weak dendritic spikes and EPSPs to action potential output. A further corollary of the theta-induced decrease of inhibition would be a degraded output precision, since in addition to strong dendritic spikes, now temporally more variable EPSPs and weak dendritic spikes contribute BMS-777607 datasheet to action potential output. This observation implies that during theta activity of CA1 ensembles more branches can efficiently contribute to output. The increase in output probability caused by a decrease in recurrent inhibition

is consistent with the elevated firing frequency of the CA1 ensemble during exploration observed in vivo (Thompson and Best, 1989). Our data suggest that the perforant path input is less strongly regulated by recurrent inhibition, when compared to Schaffer collateral input. However, repetitive CA1 pyramidal neuron activity at theta frequency resulted in a mildly augmented local inhibition observed with two-photon calcium imaging. Therefore, the proper integration of both, perforant path and Schaffer collateral excitatory inputs during theta activity, which is thought to be critically relevant for long-term spatial memory consolidation for (Remondes and

Schuman, 2002, 2004), may depend on the differential recruitment of recurrent interneurons (Pouille and Scanziani, 2004). Taken together, the input to output coupling of CA1 pyramidal neurons is controlled by an activity-dependent regulation of recurrent inhibition, which may be observed during different network activities in vivo. Furthermore we identify a mechanism, by which correlated input on dendritic branches could resist inhibitory control, which is widely independent of the network activity patterns: In both, periods of sparse and periods of higher rhythmic network activity, the generation and plasticity of dendritic spikes may serve to reliably couple the information conveyed by the activity of a presynaptic cell assembly to precise CA1 action potential output. Male Wistar rats (P21–P28, Charles River, Germany) were deeply anesthetized with an injection of ketamine (100 mg/kg, Pfizer, Germany) and xylazine (15 mg/kg; Bayer, Leverkusen, Germany) and then decapitated.

The ionic mechanisms underlying this offset firing were investigated by studying the conductances activated around resting membrane potentials (RMP: −59.9 ± 0.9mV; n = 82). A general characterization of the SPN neurons under voltage clamp in vitro showed that they possessed large sustained outward potassium currents in response to depolarization, including tetraethyl ammonium-sensitive Kv3 high voltage-activated currents and low voltage-activated, dendrotoxin-I sensitive Kv1 currents. Under current clamp SPN neurons had rapid time-course overshooting

APs (absolute amplitude: 12.1 ± 1.5mV; half-width: 0.36 ± 0.02 ms, n = 72, see also Figure S1, available online). Sound-evoked firing in neurons of the MNTB and SPN show a reciprocal relationship. Presentation of a contralateral Venetoclax solubility dmso Bcl-2 inhibition pure-tone stimulus at the characteristic frequency (CF) for an MNTB neuron gives continuous high-frequency AP firing for the duration of the stimulation, but firing is suppressed below spontaneous

activity after the end of the sound (Figure 1D). MNTB AP firing exceeds spontaneous levels as sound intensity (0 to 80 dB SPL) passes threshold and monotonically increases until reaching a plateau firing rate (Figure 1D). The opposite occurs in the SPN; sound stimulation suppresses all firing during the sound but triggers offset firing after cessation of the sound (Figure 1E) with the number of APs continuously increasing with sound intensity (beyond threshold). Stimulation of the MNTB activated endogenous inhibition in SPN neurons in vitro, followed by offset APs (100 Hz train for 100 ms, Figure 1H, upper trace, see also Figure S2). Thus, acoustic stimulation, hyperpolarizing current injection, and electrically evoked IPSPs all resulted in CYTH4 similar offset

firing. These results confirm the uniformity of evoked offset firing in both in vivo and in vitro recordings and support the use of in vitro methods to identify the ionic basis of SPN offset firing. IPSCs triggered by MNTB stimulation are blocked by the glycine receptor antagonist strychnine (1 μM), confirming the origin and transmitter of this inhibitory synaptic projection (Figure 1G). In vivo recordings confirm that evoked glycinergic IPSPs trigger offset firing but do not exclude the possibility that EPSPs might also be involved. To test this hypothesis we used repetitive IPSPs evoked by electrical stimulation of the MNTB in vitro in the presence of AMPAR and NMDAR antagonists (50 μM AP5, 10 μM CNQX). Under these conditions, well-timed offset APs were generated (Figure 1H, middle trace) as the membrane potential rapidly depolarized back to resting levels at the end of the train, thus confirming that excitatory synaptic transmission was not necessary for offset firing.

Whisker deflection was triggered by the microscope operating system, ScanImage (Pologruto et al., 2003), to allow synchronization. Custom code was used to generate a sine wave, which was then amplified and delivered to a piezo actuator. The piezoelectric stimulator was positioned approximately 5 mm from the base of the whisker. Each whisker stimulation epoch consisted of a 5 Hz, 20V signal delivered to the piezo actuator, resulting in a deflection of approximately 400 μm.

Each Selleckchem Y27632 of the five stimuli comprising the stimulus was 25 ms in duration peak-to-peak. In each imaging trial there were ten epochs of 5 Hz whisker stimulation, each 10 s apart. Neurons were distinguished from astrocytes using Sulforhodamine coinjection (Nimmerjahn et al., 2004). Analysis of the data was similar to (Mrsic-Flogel et al., 2007). All neurons in a field of view were identified using a semiautomated custom made routines (Matlab). In each trial, Olaparib nmr the fluorescence observed during a 2 s time prior to the stimulation was defined as a baseline (F0). The change of fluorescence in each frame (F) from baseline was calculated as: (F − F0)/F0. Then, the averaged trace of all ten trials was calculated. A response window was defined from the initiation of the stimulation until 1 s post. Response to a single trial was defined by three parameters:

(1) a fluorescent change of at least 5% above the baseline preceding this trial that corresponds to one spike (Ch’ng and Reid, 2010); (2) a fluorescent change greater than the mean plus three SDs calculated from a baseline derived from the 2 s preceding each of the ten trials (this baseline was computed from the median of each time point across all ten trials to reduce the effect of spontaneous spikes during baseline); and (3) at least a 3% increase in fluorescence from the former Metalloexopeptidase frame to the peak-response frame in the 2 s response window to reflect fast rise time of the signal (Greenberg et al., 2008). Responsive neurons across trials were defined based on two measure of spontaneous

activity. Statistical analysis consisted of the following: t test (Figures 1C and 1D), one-way ANOVA, Tukey’s post-hoc test (Figures 2A–2C), Mann-Whitney nonparametric test (Figures 3C, 3D, 5A–5G, 6A, 6B, 7A–7D, and 7F), and two-way ANOVA (Figures 3E and 6C training × distance), (Figures 5F and 7E training × fidelity). This work was funded by grants from the National Institute of Mental Health to J.T.T. (P50MH077972 and R01MH082935), M.S.F. (R01MH062122), and A.J.S. (P50MH077972). We thank M. Stryker, D. Buonomano, and A. Matynia for their helpful comments on earlier versions of the manuscript; R. Edelshtein for help with video editing; J. Friedman, B. Jayaprakash, and G. Arom for help with electronics; and G.W. Byeon, A. Pyo, W. Columna, and G. Evans for help in behavior.

, 2010; Yang et al., 2012), suggesting an alteration in the set-point for bidirectional

Hebbian synaptic plasticity (Cho and Bear, 2010). The same analysis was not conducted in other mutant lines (Peça et al., 2011; Wang et al., 2011). Collectively, these data support circuit defects mediated by glutamate receptors in Shank3 mutant mice that appear to be both synapse and mutation specific. It is not yet clear whether there are common core synaptic FG-4592 defects in the various mutant mice, but the phenotypic heterogeneity itself appears consistent with the clinical heterogeneity of patients harboring SHANK3 mutations. Since different mutations affect different isoforms of Shank3, some of the observed phenotypes may arise from isoform-specific effects on synaptic transmission. Firm conclusions in this regard are complicated by the fact that the different Shank3 isoforms are probably expressed in various Shank3 mutant mice, which were analyzed at different ages using slightly different protocols. Moreover, acute knockdown of Shank3 in cultured neurons decreases mGluR-dependent plasticity ( Verpelli et al., 2011), suggesting differences in effects of Shank3 on mGluR1/5 signaling over development and pointing to the need for cautious interpretation regarding the pathogenic versus compensatory roles of synaptic and circuit phenotypes observed in Shank3 mutant mice. Based on the strong genetic evidence for SHANK3

defects as a cause of human ASD, Shank3 mutant mice offer an opportunity to model autism-like behaviors in rodents. Extensive behavioral analyses were performed in Shank3 Δex4–9B(+/−,−/−) ( Bozdagi et al., Integrase inhibitor 2010; Yang et al., 2012), Δex4–9J−/− ( Wang et al., 2011), Δex4–7−/−, and Δex13–16−/− ( Peça et al., 2011) mutant mice at different ages, on different genetic backgrounds, and using different protocols. The most notable

and consistent observation was reduced social interaction and affiliation behaviors using different testing methods ( Bozdagi et al., 2010; Peça et al., 2011; Wang et al., 2011; Yang et al., 2012). Variable performances were noted in different cohorts of Δe4–9B−/− mice ( Yang et al., 2012). Repetitive behaviors measured by increased self-grooming in the home cage and behavioral inflexibility in the reverse Morris water maze were Mannose-binding protein-associated serine protease observed in Δex4–9J−/− ( Wang et al., 2011) and Δex4–9B−/− mice ( Bozdagi et al., 2010; Yang et al., 2012) but were not apparent in Δex4–7−/− mice ( Peça et al., 2011). A more marked increase in self-grooming and self-injurious behaviors was observed in Δex11−/− and Δex13–16−/− mice ( Peça et al., 2011; Schmeisser et al., 2012). Different severity of similar behaviors with different mutations may reflect Shank3 isoform-specific contributions to specific behaviors. The number, frequency, and duration of ultrasonic vocalizations were altered in a sex-specific manner in Δex4–9J−/− mice ( Wang et al., 2011).

We therefore decided to test the hypothesis that the three VGLUT isoforms confer specific properties of glutamatergic neurotransmission upon the synapses at which they are present. We used whole-cell voltage clamp to record synaptic currents from primary cultured neurons expressing different endogenous or virally expressed VGLUT isoforms and measured basic parameters of synaptic function. Our results demonstrate that expression of any VGLUT, including VGLUT3, gives a neuron the ability to release glutamate and that neurons expressing VGLUT1 exhibit lower vesicular release probability (Pvr) and altered short-term plasticity compared to VGLUT2- or VGLUT3-expressing neurons. In exploring the mechanism

by which VGLUT isoforms regulate exocytosis, we identified endophilin click here A1 as a positive regulator of release efficiency and propose that VGLUT1′s effects result from binding and inhibiting endophilin A1. We wanted to directly

compare the basic functions of VGLUT1, VGLUT2 and VGLUT3 in an otherwise identical cellular environment. Previous studies demonstrated that hippocampal VGLUT1−/− neurons and thalamic VGLUT2−/− neurons have very low or undetectable levels of VGLUT protein, virtually Inhibitor Library clinical trial no evoked or spontaneous glutamate release, and a very small readily releasable pool (RRP) of filled synaptic vesicles ( Fremeau et al., 2004, Moechars et al., 2006 and Wojcik et al., 2004). We prepared primary autaptic cultures of these neurons and used lentiviruses to induce expression of each of the three VGLUT isoforms. We then performed whole-cell voltage-clamp analysis to test for rescue of the synaptic response. Evoked responses were measured in knockout neurons and neurons infected with VGLUT1, VGLUT2, and VGLUT3-expressing lentiviruses. Expression of all three isoforms rescued the deficit in EPSC peak amplitude and EPSC charge in both VGLUT1−/− hippocampal neurons ( Figures 1A and 1D) and VGLUT2−/− thalamic neurons ( Figures 1B and 1E). The EPSC amplitudes of neurons rescued with VGLUT1, VGLUT2 and VGLUT3 were not significantly different from hippocampal VGLUT+/+ neurons infected

with a lentivirus expressing only GFP, nor were they significantly no different from each other ( Figures 1D and 1E). The charge contained in the EPSC of VGLUT1, VGLUT2 and VGLUT3 expressing hippocampal neurons were slightly larger, but not significantly different from, control neurons, and were not significantly different from each other ( Figure 1F, left panel). We also measured the size of the charge contained in the RRP by applying 500 mM sucrose ( Rosenmund and Stevens, 1996). Again all three VGLUT isoforms rescued the severe deficit seen in both VGLUT1−/− hippocampal neurons ( Figures 1C and 1F) and VGLUT2−/− thalamic neurons (data not shown) to levels not significantly different from VGLUT+/+ thalamic neurons, suggesting that the three isoforms perform the basic function of filling synaptic vesicles with glutamate in a similar manner.

To explore the dynamic range of PIRK’s effect on neuronal Dinaciclib clinical trial firing, we measured the firing frequency from each neuron over a range of current injections (0–70 pA). Cells that did not fire throughout the current range were excluded. With small current injections (<40 pA), the firing frequency decreased significantly upon UV light activation of PIRK channels (Figure 5A). With larger current injections (40–70 pA) and higher firing

frequencies, however, there was no significant change in firing frequency following UV light illumination of PIRK-expressing neurons. This ceiling effect can be explained by the native properties of strong inwardly rectifying Kir2.1 channels, which conduct little outward current at positive membrane potentials (Ishii et al., 1994 and Kubo et al., 1993). Kir channels are well known for their selleck inhibitor ability to hyperpolarize membranes and increase the threshold for firing an action potential. To examine this, we measured the minimum amount of current required to

evoke an action potential (referred to as rheobase). The rheobase increased in PIRK-expressing neurons following UV light exposure (Figure 5C). UV light activation of PIRK also hyperpolarized the resting membrane potential of PIRK-expressing neurons by −17 mV, whereas UV light had no effect on the resting potential of control neurons (Figure 5D; Figure S4G). Having successfully expressed PIRK channels in dissociated hippocampal neurons, we next attempted to express PIRK channels in vivo. Genetically encoding Uaas using orthogonal tRNA/synthetase has great potential

to address challenging biological questions in vivo, but this technology has yet to be applied in mammals. There were two main challenges for in vivo incorporation of Uaas in mammals: (1) efficient delivery and expression of the genes for the orthogonal tRNA/synthetase and the target protein into specific found tissue or cells; and (2) sufficient bioavailability of the Uaa at the target tissue and cells. We chose mouse embryos for genetically incorporating Uaas into the brain because of the ability to introduce cDNA and chemicals in utero and then to prepare brain slices pre- and postnatally (Mulder et al., 2008, Saito, 2006 and Tabata and Nakajima, 2001). We started addressing the first challenge by attempting to incorporate Leu, an endogenously available amino acid, into GFP through UAG suppression in mouse embryonic brain. The GFP_Y182  TAG reporter gene was encoded on the same plasmid with the orthogonal tRNACUALeu ( Figure 6A). Three copies of this tRNA expression cassette driven by the H1 promoter were included to increase the UAG suppression efficiency, as we previously demonstrated in mammalian cells ( Coin et al., 2011). A red fluorescent protein, mCherry, was coexpressed with the orthogonal LeuRS through the internal ribosome entry site (IRES) on the other plasmid to indicate successful gene delivery in vivo.