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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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GluN2 and GFP transgene expressionin vivomediated by adeno-associated virus. In this experiment, AAV viruses designed to express Flag-GluN2A/B, GFP, and Flag-GluN2A/BΔC from a CMV, EFS, TRE3G or 0.4αCaMKII promoter, as indicated, were infused into the basolateral complex of the amygdala (BLA). All viruses were infused into the rat BLA, except for the TRE3G promoter containing viruses – these viruses were infused into the BLA of αCaMKII-tTA transgenic mice. Twenty one days following viral infusion, coronal sections were prepared that contained the BLA and native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via immunohistochemistry(IHC) and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Viruses designed to express GFP and Flag-GluN2A/BΔC exhibited convincing and robust transgene expression in vivo. Viruses designed to express Flag-GluN2A/B were not capable of conferring GluN2 expression in vivo. *indicates virus derived from pAAV-Basic vector. Coronal sections from naïve controls were processed as negative controls for Flag-IHC and coronal sections from wild type mice were processed as controls (Control A) for mice infused with AAV-TRE3G-Flag-GluN2A.
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Fig6: GluN2 and GFP transgene expressionin vivomediated by adeno-associated virus. In this experiment, AAV viruses designed to express Flag-GluN2A/B, GFP, and Flag-GluN2A/BΔC from a CMV, EFS, TRE3G or 0.4αCaMKII promoter, as indicated, were infused into the basolateral complex of the amygdala (BLA). All viruses were infused into the rat BLA, except for the TRE3G promoter containing viruses – these viruses were infused into the BLA of αCaMKII-tTA transgenic mice. Twenty one days following viral infusion, coronal sections were prepared that contained the BLA and native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via immunohistochemistry(IHC) and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Viruses designed to express GFP and Flag-GluN2A/BΔC exhibited convincing and robust transgene expression in vivo. Viruses designed to express Flag-GluN2A/B were not capable of conferring GluN2 expression in vivo. *indicates virus derived from pAAV-Basic vector. Coronal sections from naïve controls were processed as negative controls for Flag-IHC and coronal sections from wild type mice were processed as controls (Control A) for mice infused with AAV-TRE3G-Flag-GluN2A.

Mentions: Along with testing many of these viruses in vitro, we also infused the following viruses in vivo, into the basolateral amygdala: AAV-0.4αCaMKII-GluN2A*, AAV-0.4αCaMKII-GluN2A, AAV-0.4αCaMKII-GluN2B, AAV-CMV-GluN2A, AAV-CMV-GluN2B, AAV-EFS-GluN2A, AAV-CMV-GluN2AΔC, AAV-CMV-GluN2BΔC, AAV-0.4αCaMKII-GluN2AΔC, AAV-0.4αCaMKII-GluN2BΔC, AAV-TRE3G-GluN2A, AAV-CMV-GFP, and AAV-0.4αCaMKII-GFP. Twenty one days following viral infusion, the animals were sacrificed, coronal brains sections were prepared that contained the amygdala and the tissue was processed for anti-Flag IHC or visualization of native GFP fluorescence as appropriate. All viruses were infused into rats, except the viruses containing the TRE3G promoter. These TRE3G containing viruses were infused into αCaMKII-tTA mice [38]. AAV-CMV-GluN2AΔC, AAV-CMV-GluN2BΔC, AAV-0.4αCaMKII-GluN2AΔC, AAV-0.4αCaMKII-GluN2BΔC, AAV-CMV-GFP, and AAV-0.4αCaMKII-GFP were all capable of conferring convincing transgene expression in a large number of amygdala neurons. However AAV-0.4αCaMKII-GluN2A*, AAV-0.4αCaMKII-GluN2A, AAV-0.4αCaMKII-GluN2B, AAV-CMV-GluN2A, AAV-CMV-GluN2B, AAV-EFS-GluN2A, and AAV-TRE3G-GluN2A were not capable of conferring transgene expression and these results were consistent with our in vitro findings (Figure 6).Figure 6


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)

GluN2 and GFP transgene expressionin vivomediated by adeno-associated virus. In this experiment, AAV viruses designed to express Flag-GluN2A/B, GFP, and Flag-GluN2A/BΔC from a CMV, EFS, TRE3G or 0.4αCaMKII promoter, as indicated, were infused into the basolateral complex of the amygdala (BLA). All viruses were infused into the rat BLA, except for the TRE3G promoter containing viruses – these viruses were infused into the BLA of αCaMKII-tTA transgenic mice. Twenty one days following viral infusion, coronal sections were prepared that contained the BLA and native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via immunohistochemistry(IHC) and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Viruses designed to express GFP and Flag-GluN2A/BΔC exhibited convincing and robust transgene expression in vivo. Viruses designed to express Flag-GluN2A/B were not capable of conferring GluN2 expression in vivo. *indicates virus derived from pAAV-Basic vector. Coronal sections from naïve controls were processed as negative controls for Flag-IHC and coronal sections from wild type mice were processed as controls (Control A) for mice infused with AAV-TRE3G-Flag-GluN2A.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Fig6: GluN2 and GFP transgene expressionin vivomediated by adeno-associated virus. In this experiment, AAV viruses designed to express Flag-GluN2A/B, GFP, and Flag-GluN2A/BΔC from a CMV, EFS, TRE3G or 0.4αCaMKII promoter, as indicated, were infused into the basolateral complex of the amygdala (BLA). All viruses were infused into the rat BLA, except for the TRE3G promoter containing viruses – these viruses were infused into the BLA of αCaMKII-tTA transgenic mice. Twenty one days following viral infusion, coronal sections were prepared that contained the BLA and native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via immunohistochemistry(IHC) and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Viruses designed to express GFP and Flag-GluN2A/BΔC exhibited convincing and robust transgene expression in vivo. Viruses designed to express Flag-GluN2A/B were not capable of conferring GluN2 expression in vivo. *indicates virus derived from pAAV-Basic vector. Coronal sections from naïve controls were processed as negative controls for Flag-IHC and coronal sections from wild type mice were processed as controls (Control A) for mice infused with AAV-TRE3G-Flag-GluN2A.
Mentions: Along with testing many of these viruses in vitro, we also infused the following viruses in vivo, into the basolateral amygdala: AAV-0.4αCaMKII-GluN2A*, AAV-0.4αCaMKII-GluN2A, AAV-0.4αCaMKII-GluN2B, AAV-CMV-GluN2A, AAV-CMV-GluN2B, AAV-EFS-GluN2A, AAV-CMV-GluN2AΔC, AAV-CMV-GluN2BΔC, AAV-0.4αCaMKII-GluN2AΔC, AAV-0.4αCaMKII-GluN2BΔC, AAV-TRE3G-GluN2A, AAV-CMV-GFP, and AAV-0.4αCaMKII-GFP. Twenty one days following viral infusion, the animals were sacrificed, coronal brains sections were prepared that contained the amygdala and the tissue was processed for anti-Flag IHC or visualization of native GFP fluorescence as appropriate. All viruses were infused into rats, except the viruses containing the TRE3G promoter. These TRE3G containing viruses were infused into αCaMKII-tTA mice [38]. AAV-CMV-GluN2AΔC, AAV-CMV-GluN2BΔC, AAV-0.4αCaMKII-GluN2AΔC, AAV-0.4αCaMKII-GluN2BΔC, AAV-CMV-GFP, and AAV-0.4αCaMKII-GFP were all capable of conferring convincing transgene expression in a large number of amygdala neurons. However AAV-0.4αCaMKII-GluN2A*, AAV-0.4αCaMKII-GluN2A, AAV-0.4αCaMKII-GluN2B, AAV-CMV-GluN2A, AAV-CMV-GluN2B, AAV-EFS-GluN2A, and AAV-TRE3G-GluN2A were not capable of conferring transgene expression and these results were consistent with our in vitro findings (Figure 6).Figure 6

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