Comparison of DNA Delivery and Expression Using Frequently Used Delivery Methods

Collins, Stuart G.; Morrissey, David; Rajendran, Simon; Casey, Garrett; Scallan, Martina F.; Harrison, Patrick T.; O’Sullivan, Gerald C.; Tangney, Mark

doi:10.5772/18898

Cited by 2 publications

(2 citation statements)

References 45 publications

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“…Our results on muscle function reinforce the validity of this strategy since our model reproduces the pathological features of the disease with the major protein responsible for MFM: desmin. Moreover, compared to non-viral transfection techniques such as electroporation, this method significantly improves the percentage of transfected fibers [39,40], is easier to implement in vivo and does not induce the confounding effect of muscle damage frequently observed with electroporation. In the present study, we obtained a very homogeneous expression of the mutant desmin with more than 98% of the muscle fibers being transduced and expressing the transgene.…”

Section: Discussionmentioning

confidence: 99%

Viral-mediated expression of desmin mutants to create mouse models of myofibrillar myopathy

et al. 2013

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BackgroundThe clinical features of myofibrillar myopathies display a wide phenotypic heterogeneity. To this date, no studies have evaluated this parameter due to the absence of pertinent animal models. By studying two mutants of desmin, which induce subtle phenotypic differences in patients, we address this issue using an animal model based on the use of adeno-associated virus (AAV) vectors carrying mutated desmin cDNA.MethodsAfter preparation of the vectors, they were injected directly into the tibialis anterior muscles of C57BL/6 mice to allow expression of wild-type (WT) or mutated (R406W or E413K) desmin. Measurements of maximal force were carried out on the muscle in situ and then the injected muscles were analyzed to determine the structural consequences of the desmin mutations on muscle structure (microscopic observations, histology and immunohistochemistry).ResultsInjection of AAV carrying WT desmin results in the expression of exogenous desmin in 98% of the muscle fibers without any pathological or functional perturbations. Exogenous WT and endogenous desmin are co-localized and no differences were observed compared to non-injected muscle. Expression of desmin mutants in mouse muscles induce morphological changes of muscle fibers (irregular shape and size) and the appearance of desmin accumulations around the nuclei (for R406W) or in subsarcolemmal regions of fibers (for E413K). These accumulations seem to occur and disrupt the Z-line, and a strong regeneration was observed in muscle expressing the R406W desmin, which is not the case for E413K. Moreover, both mutants of desmin studied here induce a decrease in muscle force generation capacity.ConclusionsIn this study we show that AAV-mediated expression of desmin mutants in mouse muscles recapitulate the aggregation features, the decrease in contractile function and the morphological changes observed in patients with myofibrillar myopathy. More importantly, our results suggest that the R406W desmin mutant induces a robust muscle regeneration, which is not the case for the E413K mutant. This difference could help to explain the phenotypic differences observed in patients. Our results highlight the heterogeneous pathogenic mechanisms between different desmin mutants and open the way for new advances in the study of myofibrillar myopathies.

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Section: Discussionmentioning

confidence: 99%

Viral-mediated expression of desmin mutants to create mouse models of myofibrillar myopathy

et al. 2013

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“…Transfection efficiency is typically evaluated by analyzing the expression of a luminescent protein (i.e., firefly luciferase [62]), a fluorescent protein (i.e., GFP [159]), or other easily detectable proteins such as the β-galactosidase [137], or a combination thereof [160]. Depending on the transgene delivered, the read-out system allows direct detection of the cells expressing the protein of interest, as for fluorescent proteins, through flow cytometry (FCM) and imaging techniques, or indirectly [68,137].…”

Section: Evaluation Of Transfection Effectiveness: a Trade-off Betweementioning

confidence: 99%

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

et al. 2020

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Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in vitro is strictly dependent on several experimental conditions specific to each laboratory. The lack of standard tests has thus largely contributed to the flood of inconsistent data underpinning the reproducibility crisis. A way researchers seek to address this issue is by gauging the effectiveness of newly synthesized gene delivery vectors with respect to benchmarks of seemingly well-known behavior. However, the performance of such reference molecules is also affected by the testing conditions. This survey points to non-standardized transfection settings and limited information on variables deemed relevant in this context as the major cause of such misalignments. This review provides a catalog of conditions optimized for the gold standard and internal reference, 25 kDa polyethyleneimine, that can be profitably replicated across studies for the sake of comparison. Overall, we wish to pave the way for the implementation of standardized protocols in order to make the evaluation of the effectiveness of transfectants as unbiased as possible.

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Comparison of DNA Delivery and Expression Using Frequently Used Delivery Methods

Cited by 2 publications

References 45 publications

Viral-mediated expression of desmin mutants to create mouse models of myofibrillar myopathy

Viral-mediated expression of desmin mutants to create mouse models of myofibrillar myopathy

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

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