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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 vitromediated by adeno-associated virus. (A) In this experiment, 293FT cells were transduced with AAV viruses designed to express GFP from a CMV promoter, Flag-GluN2A from the CMV, TRE3G, EFS, or RSV promoters, and Flag-GluN2B and GluN2A/BΔC from a CMV promoter. TRE3G promoter containing viruses were transduced into cells that were also transfected with the pTet-Off plasmid. Forty eight hours after viral transduction, native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via ICC and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Non-transduced cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. AAV designed to express Flag-GluN2 from the CMV, TRE3G, EFS, or RSV promoters did not confer convincing transgene expression. However AAV designed to express GFP or GluN2A/BΔC exhibited expression in > 90% of cells. (Scale bar = 20 μm) (B) Depicts similar images as depicted in A, but at lower magnification. (Scale bar = 50 μm).
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Fig5: GluN2 and GFP transgene expressionin vitromediated by adeno-associated virus. (A) In this experiment, 293FT cells were transduced with AAV viruses designed to express GFP from a CMV promoter, Flag-GluN2A from the CMV, TRE3G, EFS, or RSV promoters, and Flag-GluN2B and GluN2A/BΔC from a CMV promoter. TRE3G promoter containing viruses were transduced into cells that were also transfected with the pTet-Off plasmid. Forty eight hours after viral transduction, native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via ICC and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Non-transduced cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. AAV designed to express Flag-GluN2 from the CMV, TRE3G, EFS, or RSV promoters did not confer convincing transgene expression. However AAV designed to express GFP or GluN2A/BΔC exhibited expression in > 90% of cells. (Scale bar = 20 μm) (B) Depicts similar images as depicted in A, but at lower magnification. (Scale bar = 50 μm).

Mentions: Next we wanted to determine if the AAV viral plasmids that could confer gene expression of the GluN2 subunits could be packaged into functional virus and could be used to transduce cells grown in culture (i.e. in vitro) to induce ectopic transgene expression. AAV viruses pseudotyped as AAV2/DJ were produced using the following viral vector plasmids: EFS-GluN2A, RSV-GluN2A, TRE3G-GluN2A, CMV-GluN2A, CMV-GluN2B, CMV-GluN2AΔC, CMV-GluN2BΔC, and CMV-GFP. 293FT cells were treated with equal amounts of virus and examined for transgene expression 48 hours following application of the virus. Cells that were transduced with the TRE3G-GluN2A virus were transfected with the pTet-Off plasmid 6 hours before the virus was applied to the cells. We observed that the CMV-GFP virus which has a viral genome size of 3045 bps exhibited expression in >90% of cells. Similarly CMV-GluN2AΔC and CMV-GluN2BΔC viruses which have viral genomes sizes of 3893 bps and 3879 bps respectively, also exhibited expression in >90% of cells indicating that these viral genomes were successfully packaged into functional viral particles (Figure 5). However, AAV-CMV-GluN2A, AAV-CMV-GluN2B, AAV-TRE3G-GluN2A, AAV-EFS-GluN2A, and AAV-RSV-GluN2A, which have the viral genome sizes of, 5411, 5476, 5228, 5077, and 5055 bps respectively, demonstrated very little expression of the GluN2 transgene. Expression of the GluN2 transgene was observed in only 15–35 cells for the entire coverslip. We suspect that the lack of expression observed from AAV-CMV-GluN2A, AAV-CMV-GluN2B, AAV-TRE3G-GluN2A is likely due to the fact that these viruses were produced with viral genomes that were over the optimal packaging limit and we suspect that AAV-EFS-GluN2A, and AAV-RSV-GluN2A may not have been capable of conferring GluN2 transgene expression because these viruses were also produced with genomes above the optimal size and that the RSV and EFS promoters are not capable of conferring robust expression of the GluN2A transgene, as compared to the CMV and TRE3G promoters. AAV viruses that contain neuron specific promoters were not examined in vitro, because N2A cells are not transduced well by AAVDJ. We have examined numerous serotypes of AAV for their ability to transduce N2A cells but we have not been able to find one (data not shown).Figure 5


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 vitromediated by adeno-associated virus. (A) In this experiment, 293FT cells were transduced with AAV viruses designed to express GFP from a CMV promoter, Flag-GluN2A from the CMV, TRE3G, EFS, or RSV promoters, and Flag-GluN2B and GluN2A/BΔC from a CMV promoter. TRE3G promoter containing viruses were transduced into cells that were also transfected with the pTet-Off plasmid. Forty eight hours after viral transduction, native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via ICC and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Non-transduced cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. AAV designed to express Flag-GluN2 from the CMV, TRE3G, EFS, or RSV promoters did not confer convincing transgene expression. However AAV designed to express GFP or GluN2A/BΔC exhibited expression in > 90% of cells. (Scale bar = 20 μm) (B) Depicts similar images as depicted in A, but at lower magnification. (Scale bar = 50 μm).
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Fig5: GluN2 and GFP transgene expressionin vitromediated by adeno-associated virus. (A) In this experiment, 293FT cells were transduced with AAV viruses designed to express GFP from a CMV promoter, Flag-GluN2A from the CMV, TRE3G, EFS, or RSV promoters, and Flag-GluN2B and GluN2A/BΔC from a CMV promoter. TRE3G promoter containing viruses were transduced into cells that were also transfected with the pTet-Off plasmid. Forty eight hours after viral transduction, native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via ICC and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP or Flag-GluN2 (Texas Red) transgene expression. Non-transduced cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. AAV designed to express Flag-GluN2 from the CMV, TRE3G, EFS, or RSV promoters did not confer convincing transgene expression. However AAV designed to express GFP or GluN2A/BΔC exhibited expression in > 90% of cells. (Scale bar = 20 μm) (B) Depicts similar images as depicted in A, but at lower magnification. (Scale bar = 50 μm).
Mentions: Next we wanted to determine if the AAV viral plasmids that could confer gene expression of the GluN2 subunits could be packaged into functional virus and could be used to transduce cells grown in culture (i.e. in vitro) to induce ectopic transgene expression. AAV viruses pseudotyped as AAV2/DJ were produced using the following viral vector plasmids: EFS-GluN2A, RSV-GluN2A, TRE3G-GluN2A, CMV-GluN2A, CMV-GluN2B, CMV-GluN2AΔC, CMV-GluN2BΔC, and CMV-GFP. 293FT cells were treated with equal amounts of virus and examined for transgene expression 48 hours following application of the virus. Cells that were transduced with the TRE3G-GluN2A virus were transfected with the pTet-Off plasmid 6 hours before the virus was applied to the cells. We observed that the CMV-GFP virus which has a viral genome size of 3045 bps exhibited expression in >90% of cells. Similarly CMV-GluN2AΔC and CMV-GluN2BΔC viruses which have viral genomes sizes of 3893 bps and 3879 bps respectively, also exhibited expression in >90% of cells indicating that these viral genomes were successfully packaged into functional viral particles (Figure 5). However, AAV-CMV-GluN2A, AAV-CMV-GluN2B, AAV-TRE3G-GluN2A, AAV-EFS-GluN2A, and AAV-RSV-GluN2A, which have the viral genome sizes of, 5411, 5476, 5228, 5077, and 5055 bps respectively, demonstrated very little expression of the GluN2 transgene. Expression of the GluN2 transgene was observed in only 15–35 cells for the entire coverslip. We suspect that the lack of expression observed from AAV-CMV-GluN2A, AAV-CMV-GluN2B, AAV-TRE3G-GluN2A is likely due to the fact that these viruses were produced with viral genomes that were over the optimal packaging limit and we suspect that AAV-EFS-GluN2A, and AAV-RSV-GluN2A may not have been capable of conferring GluN2 transgene expression because these viruses were also produced with genomes above the optimal size and that the RSV and EFS promoters are not capable of conferring robust expression of the GluN2A transgene, as compared to the CMV and TRE3G promoters. AAV viruses that contain neuron specific promoters were not examined in vitro, because N2A cells are not transduced well by AAVDJ. We have examined numerous serotypes of AAV for their ability to transduce N2A cells but we have not been able to find one (data not shown).Figure 5

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