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The production of viral vectors designed to express large and difficult to express transgenes within neurons.

Holehonnur R, Lella SK, Ho A, Luong JA, Ploski JE - Mol Brain (2015)

Bottom Line: Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons.Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains.We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner.

View Article: PubMed Central - PubMed

Affiliation: School of Behavioral and Brain Sciences and the Department of Molecular & Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX, 75080, USA. roopa.hs@gmail.com.

ABSTRACT

Background: Viral vectors are frequently used to deliver and direct expression of transgenes in a spatially and temporally restricted manner within the nervous system of numerous model organisms. Despite the common use of viral vectors to direct ectopic expression of transgenes within the nervous system, creating high titer viral vectors that are capable of expressing very large transgenes or difficult to express transgenes imposes unique challenges. Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons.

Results: We created a number of custom designed AAV and lentiviral vectors that were optimized for large transgenes, by minimizing DNA sequences that were not essential, utilizing short promoter sequences of 8 widely used promoters (RSV, EFS, TRE3G, 0.4αCaMKII, 1.3αCaMKII, 0.5Synapsin, 1.1Synapsin and CMV) and utilizing a very short (~75 bps) 3' untranslated sequence. Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains.

Conclusions: We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner. Because these vectors have been optimized to accommodate large open reading frames and in some cases contain an intron to facilitate expression of difficult to express transgenes, these viral vectors likely could be useful for delivering and expressing many large or difficult to express transgenes in a neuron specific manner.

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Related in: MedlinePlus

Lentivirus designed with a 1.3αCaMKII promoter and an intron conferredin vitro and in vivoexpression of full length GluN2A. (A) Viral genome maps for pLenti7.3 vectors containing either the Flag-GluN2A coding region or a MCS with a 1.3αCaMKII promoter and intron. (B) A lentivirus plasmid designed to express Flag-GluN2A that included an intron from a 1.3αCaMKII promoter was transfected into N2A cells and 24 hours post transfection the cells were examined by ICC for Flag-GluN2 expression. This plasmid was not capable of conferring Flag-GluN2A expression. However when this plasmid was linearized, (Lenti-1.3αCaMKII + intron-GluN2A*) and then transfected as above to eliminate the RSV promoter from interfering with GluN2 expression, Flag-GluN2 expression was indeed detected. Non-transfected cells and cells transfected with the pRK5-Flag-GluN2A plasmid were processed as negative and positive ICC controls respectively (scale bar = 20 μm). (C) A lentivirus designed to express Flag-GluN2A that included an intron from a 1.3αCaMKII promoter was infused into the rat BLA. Ten days following viral infusion, coronal sections were prepared that contained the BLA and Flag-GluN2 expression was observed via IHC and fluorescence microscopy. Coronal sections from non-infused animals were processed as a negative control for Flag-IHC. (D) In this experiment, it was determined if the Flag-GluN2A expression from the 1.3αCaMKII-intron virus was predominantly restricted to neurons, by performing an IHC for Flag-GluN2A and the neuronal marker NeuN. Images depict Flag-GluN2A staining (Tx-red) with the same fields viewed for NeuN (FITC) staining and a merged image of these two images. This virus was capable of conferring GluN2A expression predominantly within neurons. Arrows point to a subset of NeuN positive cells that are also Flag-GluN2A positive (E) Quantification of data presented in (D), error bars represent the standard error of the mean, (scale bar = 50 μm).
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Fig12: Lentivirus designed with a 1.3αCaMKII promoter and an intron conferredin vitro and in vivoexpression of full length GluN2A. (A) Viral genome maps for pLenti7.3 vectors containing either the Flag-GluN2A coding region or a MCS with a 1.3αCaMKII promoter and intron. (B) A lentivirus plasmid designed to express Flag-GluN2A that included an intron from a 1.3αCaMKII promoter was transfected into N2A cells and 24 hours post transfection the cells were examined by ICC for Flag-GluN2 expression. This plasmid was not capable of conferring Flag-GluN2A expression. However when this plasmid was linearized, (Lenti-1.3αCaMKII + intron-GluN2A*) and then transfected as above to eliminate the RSV promoter from interfering with GluN2 expression, Flag-GluN2 expression was indeed detected. Non-transfected cells and cells transfected with the pRK5-Flag-GluN2A plasmid were processed as negative and positive ICC controls respectively (scale bar = 20 μm). (C) A lentivirus designed to express Flag-GluN2A that included an intron from a 1.3αCaMKII promoter was infused into the rat BLA. Ten days following viral infusion, coronal sections were prepared that contained the BLA and Flag-GluN2 expression was observed via IHC and fluorescence microscopy. Coronal sections from non-infused animals were processed as a negative control for Flag-IHC. (D) In this experiment, it was determined if the Flag-GluN2A expression from the 1.3αCaMKII-intron virus was predominantly restricted to neurons, by performing an IHC for Flag-GluN2A and the neuronal marker NeuN. Images depict Flag-GluN2A staining (Tx-red) with the same fields viewed for NeuN (FITC) staining and a merged image of these two images. This virus was capable of conferring GluN2A expression predominantly within neurons. Arrows point to a subset of NeuN positive cells that are also Flag-GluN2A positive (E) Quantification of data presented in (D), error bars represent the standard error of the mean, (scale bar = 50 μm).

Mentions: In our final experiment we reasoned that including an intron within the GluN2 transgene might enhance expression of the transgene when used with a neuron specific promoter. However to our knowledge there are no commercially available lentiviral plasmids that contain introns and this is likely because lentiviruses are RNA viruses and these introns would be spliced out during viral production. To circumvent this issue, we developed a lentiviral vector that contained our transgene expression cassette in the reverse orientation and this expression cassette also contained an intron directly downstream of the 1.3αCaMKII promoter (Figure 12). By placing the transgene expression cassette in the reverse orientation, the transgene would not be expressed during viral production and therefore the intron would not be spliced out. We transfected this plasmid into N2A cells, and examined transgene expression 24 hours post transfection. However the GluN2A transgene expression was still undetectable. We reasoned that the opposing RSV promoter could be interfering with the 1.3αCaMKII-intron-GluN2A transgene expression. In order to confirm this, we linearized the plasmid to prevent the RSV promoter from interfering with the expression from the 1.3αCaMKII promoter, and transfected it into N2A cells. Cells transfected with the linearized plasmid exhibited positive GluN2A expression (Figure 12), thereby proving our hypothesis that the RSV promoter was interfering with the expression of the opposing transgene and that the inclusion of the intronic sequence led to enhanced expression of the transgene. Next we infused this virus into the rat basolateral amygdala and 10 days later, the animals were sacrificed and the brains were sectioned in the coronal plane and the transgene expression was examined as above (Figure 12). The 1.3 αCaMKII-Intron-GluN2A transgene was appropriately expressed in neurons in vivo, underscoring that combining the neuron specific promoter with an intron, in a lentivirus could confer expression of large and difficult to express transgene in a neuronal specific manner. We have also developed this lentiviral vector with a multiple cloning site so that any transgene can be easily cloned into this vector. Collectively the results from the viral vectors created for this study are summarized in Figure 13.Figure 12


The production of viral vectors designed to express large and difficult to express transgenes within neurons.

Holehonnur R, Lella SK, Ho A, Luong JA, Ploski JE - Mol Brain (2015)

Lentivirus designed with a 1.3αCaMKII promoter and an intron conferredin vitro and in vivoexpression of full length GluN2A. (A) Viral genome maps for pLenti7.3 vectors containing either the Flag-GluN2A coding region or a MCS with a 1.3αCaMKII promoter and intron. (B) A lentivirus plasmid designed to express Flag-GluN2A that included an intron from a 1.3αCaMKII promoter was transfected into N2A cells and 24 hours post transfection the cells were examined by ICC for Flag-GluN2 expression. This plasmid was not capable of conferring Flag-GluN2A expression. However when this plasmid was linearized, (Lenti-1.3αCaMKII + intron-GluN2A*) and then transfected as above to eliminate the RSV promoter from interfering with GluN2 expression, Flag-GluN2 expression was indeed detected. Non-transfected cells and cells transfected with the pRK5-Flag-GluN2A plasmid were processed as negative and positive ICC controls respectively (scale bar = 20 μm). (C) A lentivirus designed to express Flag-GluN2A that included an intron from a 1.3αCaMKII promoter was infused into the rat BLA. Ten days following viral infusion, coronal sections were prepared that contained the BLA and Flag-GluN2 expression was observed via IHC and fluorescence microscopy. Coronal sections from non-infused animals were processed as a negative control for Flag-IHC. (D) In this experiment, it was determined if the Flag-GluN2A expression from the 1.3αCaMKII-intron virus was predominantly restricted to neurons, by performing an IHC for Flag-GluN2A and the neuronal marker NeuN. Images depict Flag-GluN2A staining (Tx-red) with the same fields viewed for NeuN (FITC) staining and a merged image of these two images. This virus was capable of conferring GluN2A expression predominantly within neurons. Arrows point to a subset of NeuN positive cells that are also Flag-GluN2A positive (E) Quantification of data presented in (D), error bars represent the standard error of the mean, (scale bar = 50 μm).
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Fig12: Lentivirus designed with a 1.3αCaMKII promoter and an intron conferredin vitro and in vivoexpression of full length GluN2A. (A) Viral genome maps for pLenti7.3 vectors containing either the Flag-GluN2A coding region or a MCS with a 1.3αCaMKII promoter and intron. (B) A lentivirus plasmid designed to express Flag-GluN2A that included an intron from a 1.3αCaMKII promoter was transfected into N2A cells and 24 hours post transfection the cells were examined by ICC for Flag-GluN2 expression. This plasmid was not capable of conferring Flag-GluN2A expression. However when this plasmid was linearized, (Lenti-1.3αCaMKII + intron-GluN2A*) and then transfected as above to eliminate the RSV promoter from interfering with GluN2 expression, Flag-GluN2 expression was indeed detected. Non-transfected cells and cells transfected with the pRK5-Flag-GluN2A plasmid were processed as negative and positive ICC controls respectively (scale bar = 20 μm). (C) A lentivirus designed to express Flag-GluN2A that included an intron from a 1.3αCaMKII promoter was infused into the rat BLA. Ten days following viral infusion, coronal sections were prepared that contained the BLA and Flag-GluN2 expression was observed via IHC and fluorescence microscopy. Coronal sections from non-infused animals were processed as a negative control for Flag-IHC. (D) In this experiment, it was determined if the Flag-GluN2A expression from the 1.3αCaMKII-intron virus was predominantly restricted to neurons, by performing an IHC for Flag-GluN2A and the neuronal marker NeuN. Images depict Flag-GluN2A staining (Tx-red) with the same fields viewed for NeuN (FITC) staining and a merged image of these two images. This virus was capable of conferring GluN2A expression predominantly within neurons. Arrows point to a subset of NeuN positive cells that are also Flag-GluN2A positive (E) Quantification of data presented in (D), error bars represent the standard error of the mean, (scale bar = 50 μm).
Mentions: In our final experiment we reasoned that including an intron within the GluN2 transgene might enhance expression of the transgene when used with a neuron specific promoter. However to our knowledge there are no commercially available lentiviral plasmids that contain introns and this is likely because lentiviruses are RNA viruses and these introns would be spliced out during viral production. To circumvent this issue, we developed a lentiviral vector that contained our transgene expression cassette in the reverse orientation and this expression cassette also contained an intron directly downstream of the 1.3αCaMKII promoter (Figure 12). By placing the transgene expression cassette in the reverse orientation, the transgene would not be expressed during viral production and therefore the intron would not be spliced out. We transfected this plasmid into N2A cells, and examined transgene expression 24 hours post transfection. However the GluN2A transgene expression was still undetectable. We reasoned that the opposing RSV promoter could be interfering with the 1.3αCaMKII-intron-GluN2A transgene expression. In order to confirm this, we linearized the plasmid to prevent the RSV promoter from interfering with the expression from the 1.3αCaMKII promoter, and transfected it into N2A cells. Cells transfected with the linearized plasmid exhibited positive GluN2A expression (Figure 12), thereby proving our hypothesis that the RSV promoter was interfering with the expression of the opposing transgene and that the inclusion of the intronic sequence led to enhanced expression of the transgene. Next we infused this virus into the rat basolateral amygdala and 10 days later, the animals were sacrificed and the brains were sectioned in the coronal plane and the transgene expression was examined as above (Figure 12). The 1.3 αCaMKII-Intron-GluN2A transgene was appropriately expressed in neurons in vivo, underscoring that combining the neuron specific promoter with an intron, in a lentivirus could confer expression of large and difficult to express transgene in a neuronal specific manner. We have also developed this lentiviral vector with a multiple cloning site so that any transgene can be easily cloned into this vector. Collectively the results from the viral vectors created for this study are summarized in Figure 13.Figure 12

Bottom Line: Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons.Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains.We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner.

View Article: PubMed Central - PubMed

Affiliation: School of Behavioral and Brain Sciences and the Department of Molecular & Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX, 75080, USA. roopa.hs@gmail.com.

ABSTRACT

Background: Viral vectors are frequently used to deliver and direct expression of transgenes in a spatially and temporally restricted manner within the nervous system of numerous model organisms. Despite the common use of viral vectors to direct ectopic expression of transgenes within the nervous system, creating high titer viral vectors that are capable of expressing very large transgenes or difficult to express transgenes imposes unique challenges. Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons.

Results: We created a number of custom designed AAV and lentiviral vectors that were optimized for large transgenes, by minimizing DNA sequences that were not essential, utilizing short promoter sequences of 8 widely used promoters (RSV, EFS, TRE3G, 0.4αCaMKII, 1.3αCaMKII, 0.5Synapsin, 1.1Synapsin and CMV) and utilizing a very short (~75 bps) 3' untranslated sequence. Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains.

Conclusions: We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner. Because these vectors have been optimized to accommodate large open reading frames and in some cases contain an intron to facilitate expression of difficult to express transgenes, these viral vectors likely could be useful for delivering and expressing many large or difficult to express transgenes in a neuron specific manner.

Show MeSH
Related in: MedlinePlus