A dual promoter lentiviral vector for the in vivo evaluation of gene therapeutic approaches to axon regeneration after spinal cord injury

Löw, Karin; Blesch, Armin; Herrmann, Julia; Tuszynski, Mark H.

doi:10.1038/gt.2010.14

Cited by 21 publications

(15 citation statements)

References 65 publications

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“…This plasmid was modified by replacing the CMV promoter with the CAG composite promoter (human cytomegalovirus immediate-early enhancer with a modified chicken beta-actin promoter and beta globin intron) for the expression of EGFP (Niwa et al, 1991). The EGFP cDNA in this plasmid can be exchanged for any other transgene of interest using the Gateway (Invitrogen) recombination system (Low et al, 2010). Expression of a second reporter gene, copGFP, is driven by the EF-1α promoter.…”

Section: Methodsmentioning

confidence: 99%

“…The total size of this plasmid is approximately 10 kb. The combination of CAG and EF-1α promoters has previously been shown to allow for the simultaneous, long-term expression of two transgenes in adult CNS neurons after lentiviral vector delivery (Low et al, 2010). A lentiviral plasmid was used to allow for the rapid transition from in vitro electroporation to in vivo viral gene transfer.…”

Section: Methodsmentioning

confidence: 99%

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Optimization of adult sensory neuron electroporation to study mechanisms of neurite growth

McCall¹,

Nicholson²,

Weidner³

et al. 2012

Front. Mol. Neurosci.

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The development of eukaryotic transfection technologies has been rapid in recent years, providing the opportunity to better analyze cell-autonomous mechanisms influencing various cellular processes, including cell-intrinsic regulators of regenerative neurite growth and survival. Electroporation is one of the more effective methodologies for transfection of post-mitotic neurons demonstrating sufficient neuronal survival and transfection efficiency. To further maximize the number of transfected neurons especially with large plasmids, to limit the cellular exposure to serum, and to minimize the number of animals required for cell isolation per experiment, we compared two state-of-the-art electroporation devices for in vitro transfection of adult rat dorsal root ganglion (DRG) neuron cultures. By refining different parameters, transfection efficiencies of 39–42% could be achieved using the Lonza 4D-Nucleofector X-unit system, 1.5–2-fold higher rates than those that have been previously published for adult DRG neurons using smaller plasmid sizes. Our protocol further limits the number of cells required to 3 × 105 cells per 20 μl reaction using only 2 μg DNA/reaction and allows for the complete omission of serum post-transfection. Application of this optimized protocol will contribute to furthering the study of neuron-intrinsic mechanisms responsible for growth and survival under physiological and pathophysiological conditions.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Optimization of adult sensory neuron electroporation to study mechanisms of neurite growth

McCall¹,

Nicholson²,

Weidner³

et al. 2012

Front. Mol. Neurosci.

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show abstract

“…In this way, although individual cells will still receive varying levels of TFs, the ratio of the two will be much more consistent. Alternatively, a dual promoter design in which each TF is under the control of a different promoter, could enable skewed ratios by selecting promoters with differing levels of activity in the cell type of interest [48]. In addition, when the optimal ratio is unknown, it should also be possible to take advantage of inducible promoters to systematically vary the production of exogenously expressed TFs.…”

Section: Physical Interactionmentioning

confidence: 99%

Selecting optimal combinations of transcription factors to promote axon regeneration: Why mechanisms matter

Venkatesh

Blackmore

2017

Neuroscience Letters

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Recovery from injuries to the central nervous system, including spinal cord injury, is constrained in part by the intrinsically low ability of many CNS neurons to mount an effective regenerative growth response. To improve outcomes, it is essential to understand and ultimately reverse these neuron-intrinsic constraints. Genetic manipulation of key transcription factors (TFs), which act to orchestrate production of multiple regeneration-associated genes, has emerged as a promising strategy. It is likely that no single TF will be sufficient to fully restore neuron-intrinsic growth potential, and that multiple, functionally interacting factors will be needed. An extensive literature, mostly from non-neural cell types, has identified potential mechanisms by which TFs can functionally synergize. Here we examine four potential mechanisms of TF/TF interaction; physical interaction, transcriptional cross-regulation, signaling-based cross regulation, and co-occupancy of regulatory DNA. For each mechanism, we consider how existing knowledge can be used to guide the discovery and effective use of TF combinations in the context of regenerative neuroscience. This mechanistic insight into TF interactions is needed to accelerate the design of effective TF-based interventions to relieve neuron-intrinsic constraints to regeneration and to foster recovery from CNS injury.

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“…GFP, GFP-SnoN, or GFP-SnoN-D-box mutation plasmids were expressed by a hybrid chick beta actin – minimal CMV (CAG) promoter, and the reporter gene copGFP was driven by an EF1-α promoter, as previously described [11]. To facilitate histological detection of SnoN, we fused the GFP reporter to the amino terminal of SnoN; the resulting fusion protein was functional, as shown in Results.…”

Section: Methodsmentioning

confidence: 99%

SnoN Facilitates Axonal Regeneration after Spinal Cord Injury

2013

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Adult CNS neurons exhibit a reduced capacity for growth compared to developing neurons, due in part to downregulation of growth-associated genes as development is completed. We tested the hypothesis that SnoN, an embryonically regulated transcription factor that specifies growth of the axonal compartment, can enhance growth in injured adult neurons. In vitro, SnoN overexpression in dissociated adult DRG neuronal cultures significantly enhanced neurite outgrowth. Moreover, TGF-β1, a negative regulator of SnoN, inhibited neurite outgrowth, and SnoN over-expression overcame this inhibition. We then examined whether SnoN influenced axonal regeneration in vivo: indeed, expression of a mutant form of SnoN resistant to degradation significantly enhanced axonal regeneration following cervical spinal cord injury, despite peri-lesional upregulation of TGF-β1. Thus, a developmental mechanism that specifies extension of the axonal compartment also promotes axonal regeneration after adult CNS injury.

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A dual promoter lentiviral vector for the in vivo evaluation of gene therapeutic approaches to axon regeneration after spinal cord injury

Cited by 21 publications

References 65 publications

Optimization of adult sensory neuron electroporation to study mechanisms of neurite growth

Optimization of adult sensory neuron electroporation to study mechanisms of neurite growth

Selecting optimal combinations of transcription factors to promote axon regeneration: Why mechanisms matter

SnoN Facilitates Axonal Regeneration after Spinal Cord Injury

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