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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

The CMV, TRE3G, EFS and RSV promoters differ in their ability to confer GluN2A expressionin vitro. (A) Representative ICC images for Flag-GluN2A expression and associated GFP and DAPI staining from transfected cells. In this experiment, AAV plasmids designed to express GluN2A from one of the following promoters, (CMV, TRE3G, EFS, RSV), were cotransfected into 293FT cells with a plasmid containing a CMV-GFP transgene and the pTet-Off plasmid. Twenty four hours after transfection, 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 and Flag-GluN2 (Texas Red) transgene expression. GFP was used as a transfection efficiency control. (B) Quantitation of Flag-GluN2A expression data presented in (A). The Texas Red (Flag-GluN2A) expression levels were normalized to GFP expression levels and these data were plotted as average percent expression as compared to the control group. n = 6, Error bars represent standard error of the mean (SEM). The AAV-CMV-GluN2A plasmid conferred similar Flag-GluN2A expression as the positive control (pRK5-Flag-GluN2A) and these exhibited higher levels of Flag-GluN2A expression, as compared to TRE3G, EFS, and RSV GluN2A containing plasmids. (C)p values for one-way ANOVA with Fisher’s PLSD post-hoc test for (B). Differences were considered significant if, p < 0.05. (D) Mean OD for GFP expression and DAPI staining for Flag-GluN2A expression data presented in (A). One-way ANOVA revealed that GFP levels (F(4,25) = 1.532; p = 0.2235) and DAPI levels (F(4,25 = 1.652; p = 0.1925), did not differ significantly indicating that transfection efficiency did not differ among the groups and the number of cells quantified did not differ significantly among the groups.
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Fig4: The CMV, TRE3G, EFS and RSV promoters differ in their ability to confer GluN2A expressionin vitro. (A) Representative ICC images for Flag-GluN2A expression and associated GFP and DAPI staining from transfected cells. In this experiment, AAV plasmids designed to express GluN2A from one of the following promoters, (CMV, TRE3G, EFS, RSV), were cotransfected into 293FT cells with a plasmid containing a CMV-GFP transgene and the pTet-Off plasmid. Twenty four hours after transfection, 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 and Flag-GluN2 (Texas Red) transgene expression. GFP was used as a transfection efficiency control. (B) Quantitation of Flag-GluN2A expression data presented in (A). The Texas Red (Flag-GluN2A) expression levels were normalized to GFP expression levels and these data were plotted as average percent expression as compared to the control group. n = 6, Error bars represent standard error of the mean (SEM). The AAV-CMV-GluN2A plasmid conferred similar Flag-GluN2A expression as the positive control (pRK5-Flag-GluN2A) and these exhibited higher levels of Flag-GluN2A expression, as compared to TRE3G, EFS, and RSV GluN2A containing plasmids. (C)p values for one-way ANOVA with Fisher’s PLSD post-hoc test for (B). Differences were considered significant if, p < 0.05. (D) Mean OD for GFP expression and DAPI staining for Flag-GluN2A expression data presented in (A). One-way ANOVA revealed that GFP levels (F(4,25) = 1.532; p = 0.2235) and DAPI levels (F(4,25 = 1.652; p = 0.1925), did not differ significantly indicating that transfection efficiency did not differ among the groups and the number of cells quantified did not differ significantly among the groups.

Mentions: We were interested in developing viruses that could deliver GluN2A or GluN2B subunit transgenes into brain cells and express these transgenes specifically within neurons. Here we describe developing AAV for this purpose. Because AAV has an optimal packaging limit of ~4.7 - 5.0 kb we attempted to minimize the transgene sizes as much as possible. The first viral vectors created were done so using a commercially available AAV2 genome plasmid (pAAV-Basic, Vector Biolabs) and these vectors were designed to express the following transgenes: Flag-GluN2A, Flag-GluN2B, Green fluorescent protein (GFP), C-terminal deletion mutant of Flag-GluN2A (Flag-GluN2AΔC) and a C-terminal deletion mutant of Flag-GluN2B (Flag-GluN2BΔC). These transgenes were designed with the short version of the αCaMKII promoter (0.4), and ~200 bps 3′ UTR that contained an SV40 based polyadenylation signal sequence. Versions of the Flag-GluN2AΔC and Flag-GluN2BΔC plasmids were also created that contained a cytomegalovirus (CMV) promoter instead of the 0.4αCaMKII promoter. These plasmids were transfected into Neuro 2A(N2A) cells and 24 hours later anti-Flag immunocytochemistry (ICC) was performed to detect the ectopic expression of the Flag-tagged GluN2 transgenes and the native GFP fluorescence for the GFP transgene was detected using standard fluorescence microscopy. Transgene expression was detected for 0.4αCaMKII-GFP, 0.4αCaMKII-Flag-GluN2AΔC, 0.4αCaMKII-Flag-GluN2BΔC, CMV-Flag-GluN2AΔC, and CMV-Flag-GluN2BΔC plasmids. In contrast we were not able to detect expression of 0.4αCaMKII-Flag-GluN2A and 0.4αCaMKII-Flag-GluN2B. Since each of the 0.4αCaMKII promoter containing plasmids were essentially identical by design, these data indicated that 0.4αCaMKII promoter could confer gene expression within this cell line, but the cytoplasmic C-terminal domains of GluN2A and GluN2B must interfere with the expression of these transgenes or the stability of GluN2 gene products (Figure 1). Each of the viral vector plasmids produced using pAAV-Basic will be denoted with an asterisk(*) to differentiate them from other vectors. Next, new viral vector plasmids were generated using a different AAV2 genome plasmid ([31], see Methods), because it allowed the transgenes to be ligated directly adjacent to the ITRs, which eliminated 145 bps of unnecessary DNA compared to the pAAV-Basic vector. In these newer versions of viral vectors, a shorter ~75 bps 3′UTR was used, which contained an SV40 based polyadenylation signal sequence – this further increased the available space that could be used for potential transgene DNA, effectively increasing the size of transgenes that could be delivered by AAV (Figure 2). The Flag-GluN2A, Flag-GluN2B, and GFP coding regions were cloned into these vectors and subsequently versions of these plasmids were generated to contain one of the following different promoters: 0.4αCaMKII, 1.3αCaMKII, 0.5Synapsin, 1.1Synapsin, Cytomegalovirus (CMV), Short form of Elongation Factor-1α (EFS), Rous Sarcoma Virus (RSV) and third generation Tet-inducible promoter (TRE3G). The plasmids with neuron specific promoters (i.e. CaMKII, Synapsin) were transfected into N2A cells, and the other plasmids were transfected into 293FT cells. The plasmids containing the TRE3G promoter were cotransfected with the pTet-Off plasmid (Clontech), containing the tetracycline transactivator (tTA) transgene, which is necessary to confer expression from the TRE3G promoter. Transgene expression from each of the AAV plasmids was examined 24 hours post transfection. GFP was expressed robustly by each promoter, but GluN2A and GluN2B were only expressed by the CMV, EFS, RSV, and TRE3G promoters (Figure 3). Notably none of the plasmids with neuron specific promoters (i.e. synapsin, CaMKII) were capable of conferring detectable expression of the full length Flag GluN2 transgenes and the CMV, TRE3G, EFS, and RSV promoters exhibited a differential ability to drive detectable expression of the GluN2 transgenes, where the CMV and TRE3G promoters induced the best expression and the RSV and EFS promoters comparatively induced less expression (Figure 4). Because these vectors have been optimized to minimize unnecessary DNA sequences, and to contain short promoters and 3′UTR sequences, these vectors likely could be useful for the expression of many large transgenes. GFP or GluN2 coding regions can be excised from these vectors and other transgenes can easily be inserted into these vectors.Figure 1


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)

The CMV, TRE3G, EFS and RSV promoters differ in their ability to confer GluN2A expressionin vitro. (A) Representative ICC images for Flag-GluN2A expression and associated GFP and DAPI staining from transfected cells. In this experiment, AAV plasmids designed to express GluN2A from one of the following promoters, (CMV, TRE3G, EFS, RSV), were cotransfected into 293FT cells with a plasmid containing a CMV-GFP transgene and the pTet-Off plasmid. Twenty four hours after transfection, 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 and Flag-GluN2 (Texas Red) transgene expression. GFP was used as a transfection efficiency control. (B) Quantitation of Flag-GluN2A expression data presented in (A). The Texas Red (Flag-GluN2A) expression levels were normalized to GFP expression levels and these data were plotted as average percent expression as compared to the control group. n = 6, Error bars represent standard error of the mean (SEM). The AAV-CMV-GluN2A plasmid conferred similar Flag-GluN2A expression as the positive control (pRK5-Flag-GluN2A) and these exhibited higher levels of Flag-GluN2A expression, as compared to TRE3G, EFS, and RSV GluN2A containing plasmids. (C)p values for one-way ANOVA with Fisher’s PLSD post-hoc test for (B). Differences were considered significant if, p < 0.05. (D) Mean OD for GFP expression and DAPI staining for Flag-GluN2A expression data presented in (A). One-way ANOVA revealed that GFP levels (F(4,25) = 1.532; p = 0.2235) and DAPI levels (F(4,25 = 1.652; p = 0.1925), did not differ significantly indicating that transfection efficiency did not differ among the groups and the number of cells quantified did not differ significantly among the groups.
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Fig4: The CMV, TRE3G, EFS and RSV promoters differ in their ability to confer GluN2A expressionin vitro. (A) Representative ICC images for Flag-GluN2A expression and associated GFP and DAPI staining from transfected cells. In this experiment, AAV plasmids designed to express GluN2A from one of the following promoters, (CMV, TRE3G, EFS, RSV), were cotransfected into 293FT cells with a plasmid containing a CMV-GFP transgene and the pTet-Off plasmid. Twenty four hours after transfection, 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 and Flag-GluN2 (Texas Red) transgene expression. GFP was used as a transfection efficiency control. (B) Quantitation of Flag-GluN2A expression data presented in (A). The Texas Red (Flag-GluN2A) expression levels were normalized to GFP expression levels and these data were plotted as average percent expression as compared to the control group. n = 6, Error bars represent standard error of the mean (SEM). The AAV-CMV-GluN2A plasmid conferred similar Flag-GluN2A expression as the positive control (pRK5-Flag-GluN2A) and these exhibited higher levels of Flag-GluN2A expression, as compared to TRE3G, EFS, and RSV GluN2A containing plasmids. (C)p values for one-way ANOVA with Fisher’s PLSD post-hoc test for (B). Differences were considered significant if, p < 0.05. (D) Mean OD for GFP expression and DAPI staining for Flag-GluN2A expression data presented in (A). One-way ANOVA revealed that GFP levels (F(4,25) = 1.532; p = 0.2235) and DAPI levels (F(4,25 = 1.652; p = 0.1925), did not differ significantly indicating that transfection efficiency did not differ among the groups and the number of cells quantified did not differ significantly among the groups.
Mentions: We were interested in developing viruses that could deliver GluN2A or GluN2B subunit transgenes into brain cells and express these transgenes specifically within neurons. Here we describe developing AAV for this purpose. Because AAV has an optimal packaging limit of ~4.7 - 5.0 kb we attempted to minimize the transgene sizes as much as possible. The first viral vectors created were done so using a commercially available AAV2 genome plasmid (pAAV-Basic, Vector Biolabs) and these vectors were designed to express the following transgenes: Flag-GluN2A, Flag-GluN2B, Green fluorescent protein (GFP), C-terminal deletion mutant of Flag-GluN2A (Flag-GluN2AΔC) and a C-terminal deletion mutant of Flag-GluN2B (Flag-GluN2BΔC). These transgenes were designed with the short version of the αCaMKII promoter (0.4), and ~200 bps 3′ UTR that contained an SV40 based polyadenylation signal sequence. Versions of the Flag-GluN2AΔC and Flag-GluN2BΔC plasmids were also created that contained a cytomegalovirus (CMV) promoter instead of the 0.4αCaMKII promoter. These plasmids were transfected into Neuro 2A(N2A) cells and 24 hours later anti-Flag immunocytochemistry (ICC) was performed to detect the ectopic expression of the Flag-tagged GluN2 transgenes and the native GFP fluorescence for the GFP transgene was detected using standard fluorescence microscopy. Transgene expression was detected for 0.4αCaMKII-GFP, 0.4αCaMKII-Flag-GluN2AΔC, 0.4αCaMKII-Flag-GluN2BΔC, CMV-Flag-GluN2AΔC, and CMV-Flag-GluN2BΔC plasmids. In contrast we were not able to detect expression of 0.4αCaMKII-Flag-GluN2A and 0.4αCaMKII-Flag-GluN2B. Since each of the 0.4αCaMKII promoter containing plasmids were essentially identical by design, these data indicated that 0.4αCaMKII promoter could confer gene expression within this cell line, but the cytoplasmic C-terminal domains of GluN2A and GluN2B must interfere with the expression of these transgenes or the stability of GluN2 gene products (Figure 1). Each of the viral vector plasmids produced using pAAV-Basic will be denoted with an asterisk(*) to differentiate them from other vectors. Next, new viral vector plasmids were generated using a different AAV2 genome plasmid ([31], see Methods), because it allowed the transgenes to be ligated directly adjacent to the ITRs, which eliminated 145 bps of unnecessary DNA compared to the pAAV-Basic vector. In these newer versions of viral vectors, a shorter ~75 bps 3′UTR was used, which contained an SV40 based polyadenylation signal sequence – this further increased the available space that could be used for potential transgene DNA, effectively increasing the size of transgenes that could be delivered by AAV (Figure 2). The Flag-GluN2A, Flag-GluN2B, and GFP coding regions were cloned into these vectors and subsequently versions of these plasmids were generated to contain one of the following different promoters: 0.4αCaMKII, 1.3αCaMKII, 0.5Synapsin, 1.1Synapsin, Cytomegalovirus (CMV), Short form of Elongation Factor-1α (EFS), Rous Sarcoma Virus (RSV) and third generation Tet-inducible promoter (TRE3G). The plasmids with neuron specific promoters (i.e. CaMKII, Synapsin) were transfected into N2A cells, and the other plasmids were transfected into 293FT cells. The plasmids containing the TRE3G promoter were cotransfected with the pTet-Off plasmid (Clontech), containing the tetracycline transactivator (tTA) transgene, which is necessary to confer expression from the TRE3G promoter. Transgene expression from each of the AAV plasmids was examined 24 hours post transfection. GFP was expressed robustly by each promoter, but GluN2A and GluN2B were only expressed by the CMV, EFS, RSV, and TRE3G promoters (Figure 3). Notably none of the plasmids with neuron specific promoters (i.e. synapsin, CaMKII) were capable of conferring detectable expression of the full length Flag GluN2 transgenes and the CMV, TRE3G, EFS, and RSV promoters exhibited a differential ability to drive detectable expression of the GluN2 transgenes, where the CMV and TRE3G promoters induced the best expression and the RSV and EFS promoters comparatively induced less expression (Figure 4). Because these vectors have been optimized to minimize unnecessary DNA sequences, and to contain short promoters and 3′UTR sequences, these vectors likely could be useful for the expression of many large transgenes. GFP or GluN2 coding regions can be excised from these vectors and other transgenes can easily be inserted into these vectors.Figure 1

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