Limits...
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.

Show MeSH

Related in: MedlinePlus

GluN2 and GFP transgene expressionin vitrofrom lentiviral vector plasmids. Representative ICC images from transfections with lentiviral plasmids designed to express Flag-GluN2A/B or GFP transgenes utilizing one of the following promoters: 1.3αCaMKII, 0.4αCaMKII, 1.1Synapsin, 0.5Synapsin, CMV, or TRE3G. Plasmids containing transgenes controlled by the neuron specific, 0.4αCaMKII, 1.3αCaMKII, 0.5synapsin and 1.1synapsin promoters were transfected into N2A cells. Plasmids containing transgenes controlled by CMV and TRE3G promoters were transfected into 293FT cells. TRE3G promoter containing plasmids were co-transfected with 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 or Flag-GluN2 (Texas Red) transgene expression. All lentiviral plasmids were capable of conferring GluN2 or GFP expression as intended. Untransfected cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. (Scale bar = 20 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4359567&req=5

Fig8: GluN2 and GFP transgene expressionin vitrofrom lentiviral vector plasmids. Representative ICC images from transfections with lentiviral plasmids designed to express Flag-GluN2A/B or GFP transgenes utilizing one of the following promoters: 1.3αCaMKII, 0.4αCaMKII, 1.1Synapsin, 0.5Synapsin, CMV, or TRE3G. Plasmids containing transgenes controlled by the neuron specific, 0.4αCaMKII, 1.3αCaMKII, 0.5synapsin and 1.1synapsin promoters were transfected into N2A cells. Plasmids containing transgenes controlled by CMV and TRE3G promoters were transfected into 293FT cells. TRE3G promoter containing plasmids were co-transfected with 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 or Flag-GluN2 (Texas Red) transgene expression. All lentiviral plasmids were capable of conferring GluN2 or GFP expression as intended. Untransfected cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. (Scale bar = 20 μm).

Mentions: Because we were unable to develop AAV that could deliver and express the full length GluN2 transgenes effectively, we sought to develop a lentiviral vector that could deliver the GluN2 transgenes to neurons in vivo to confer ectopic expression of these transgenes. We started by modifying the pLenti.7.3 DEST vector (Invitrogen), by removing the emerald green fluorescent protein (emGFP) expression cassette and removing the DNA between the beginning of the CMV promoter to the beginning of the WPRE site and cloning in a multiple cloning site (MCS). This was performed to facilitate the cloning of transgenes of interest and to remove unessential DNA from the lentiviral genome to reduce the overall size of the viral genome as much as possible. The Flag-GluN2A, Flag-GluN2B, and GFP coding regions were cloned into this custom modified lenti vector and subsequently versions of these plasmids were generated to contain one of the following different promoters: 0.4αCaMKII, 1.3αCaMKII, 1.1Synapsin, 0.5Synapsin, CMV and TRE3G (Figure 7). These plasmids were transfected into cells lines, and the expression of the transgene was examined 24 hours post transfection as described above. The plasmids containing neuron specific promoters were transfected into N2A cells and the remaining plasmids were transfected into 293FT cells. Each of these plasmids exhibited transgene expression (Figure 8). This result was surprising since the neuron specific promoters were not capable of conferring detectable expression of full length GluN2 transgene expression when these transgenes were in the AAV vector plasmids. Additionally when the lentiviral plasmids that contained neuron specific promoters were transfected into 293FT cells, transgene expression was detected; however these neuron specific promoters do not confer appreciable transcriptional activation in this cell line. We hypothesized that the RSV promoter which is present at the beginning of the lentiviral genome plasmid, but not present in the lentivirus, could be responsible for this cryptic expression. To test this hypothesis we generated the lenti viral plasmid 0.4αCaMKII-GluN2A without the RSV promoter and transfected this plasmid into 293FT cells along with the unmodified version of the plasmid. Twenty four hours post transfection, the expression levels of GluNA were examined, and as expected, GluN2A transgene expression was not detected from cells transfected with the plasmid without the RSV promoter, indicating it was the RSV promoter that was responsible for this spurious transgene expression (Figure 9).Figure 7


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 vitrofrom lentiviral vector plasmids. Representative ICC images from transfections with lentiviral plasmids designed to express Flag-GluN2A/B or GFP transgenes utilizing one of the following promoters: 1.3αCaMKII, 0.4αCaMKII, 1.1Synapsin, 0.5Synapsin, CMV, or TRE3G. Plasmids containing transgenes controlled by the neuron specific, 0.4αCaMKII, 1.3αCaMKII, 0.5synapsin and 1.1synapsin promoters were transfected into N2A cells. Plasmids containing transgenes controlled by CMV and TRE3G promoters were transfected into 293FT cells. TRE3G promoter containing plasmids were co-transfected with 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 or Flag-GluN2 (Texas Red) transgene expression. All lentiviral plasmids were capable of conferring GluN2 or GFP expression as intended. Untransfected cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. (Scale bar = 20 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359567&req=5

Fig8: GluN2 and GFP transgene expressionin vitrofrom lentiviral vector plasmids. Representative ICC images from transfections with lentiviral plasmids designed to express Flag-GluN2A/B or GFP transgenes utilizing one of the following promoters: 1.3αCaMKII, 0.4αCaMKII, 1.1Synapsin, 0.5Synapsin, CMV, or TRE3G. Plasmids containing transgenes controlled by the neuron specific, 0.4αCaMKII, 1.3αCaMKII, 0.5synapsin and 1.1synapsin promoters were transfected into N2A cells. Plasmids containing transgenes controlled by CMV and TRE3G promoters were transfected into 293FT cells. TRE3G promoter containing plasmids were co-transfected with 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 or Flag-GluN2 (Texas Red) transgene expression. All lentiviral plasmids were capable of conferring GluN2 or GFP expression as intended. Untransfected cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. (Scale bar = 20 μm).
Mentions: Because we were unable to develop AAV that could deliver and express the full length GluN2 transgenes effectively, we sought to develop a lentiviral vector that could deliver the GluN2 transgenes to neurons in vivo to confer ectopic expression of these transgenes. We started by modifying the pLenti.7.3 DEST vector (Invitrogen), by removing the emerald green fluorescent protein (emGFP) expression cassette and removing the DNA between the beginning of the CMV promoter to the beginning of the WPRE site and cloning in a multiple cloning site (MCS). This was performed to facilitate the cloning of transgenes of interest and to remove unessential DNA from the lentiviral genome to reduce the overall size of the viral genome as much as possible. The Flag-GluN2A, Flag-GluN2B, and GFP coding regions were cloned into this custom modified lenti vector and subsequently versions of these plasmids were generated to contain one of the following different promoters: 0.4αCaMKII, 1.3αCaMKII, 1.1Synapsin, 0.5Synapsin, CMV and TRE3G (Figure 7). These plasmids were transfected into cells lines, and the expression of the transgene was examined 24 hours post transfection as described above. The plasmids containing neuron specific promoters were transfected into N2A cells and the remaining plasmids were transfected into 293FT cells. Each of these plasmids exhibited transgene expression (Figure 8). This result was surprising since the neuron specific promoters were not capable of conferring detectable expression of full length GluN2 transgene expression when these transgenes were in the AAV vector plasmids. Additionally when the lentiviral plasmids that contained neuron specific promoters were transfected into 293FT cells, transgene expression was detected; however these neuron specific promoters do not confer appreciable transcriptional activation in this cell line. We hypothesized that the RSV promoter which is present at the beginning of the lentiviral genome plasmid, but not present in the lentivirus, could be responsible for this cryptic expression. To test this hypothesis we generated the lenti viral plasmid 0.4αCaMKII-GluN2A without the RSV promoter and transfected this plasmid into 293FT cells along with the unmodified version of the plasmid. Twenty four hours post transfection, the expression levels of GluNA were examined, and as expected, GluN2A transgene expression was not detected from cells transfected with the plasmid without the RSV promoter, indicating it was the RSV promoter that was responsible for this spurious transgene expression (Figure 9).Figure 7

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