isolation-culture-and-transduction-of-adult-mouse-cardiomyocytes

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

University of California, San Francisco

Mustard oil [allyl isothiocyanate (AITC)] and cinnamaldehyde (CA), agonists of the ion channel TRPA1 expressed in sensory neurons, elicit a burning sensation and heat hyperalgesia. We tested whether these phenomena are reflected in the responses of lumbar spinal wide-dynamic range (WDR) neurons recorded in pentobarbital-anesthetized rats. Responses to electrical and graded mechanical and noxious thermal stimulation were tested before and after cutaneous application of AITC or CA. Repetitive application of AITC initially increased the firing rate of 52% of units followed by rapid desensitization that persisted when AITC was reapplied 30 min later. Responses to noxious thermal, but not mechanical, stimuli were significantly enhanced irrespective of whether the neuron was directly activated by AITC. Windup elicited by percutaneous or sciatic nerve electrical stimulation was significantly reduced post-AITC. These results indicate that AITC produced central inhibition and peripheral sensitization of heat nociceptors. CA did not directly excite WDR neurons, and significantly enhanced responses to noxious heat while not affecting windup or responses to skin cooling or mechanical stimulation, indicating a peripheral sensitization of heat nociceptors.

Effects of TRPA1 agonists mustard oil and cinnamaldehyde on lumbar spinal wide-dynamic range neuronal responses to innocuous and noxious cutaneous stimuli in rats.

The extent to which thermostability influences the location of protein fragmentation sites that allow retention of function is not known. To evaluate this, we used a novel transposase-based approach to create libraries of vectors that express structurally-related fragments of Bacillus subtilis adenylate kinase (BsAK) and Thermotoga neapolitana adenylate kinase (TnAK) with identical modifications at their termini, and we selected for variants in each library that complement the growth of Escherichia coli with a temperature-sensitive adenylate kinase (AK). Mutants created using the hyperthermophilic TnAK were found to support growth with a higher frequency (44%) than those generated from the mesophilic BsAK (6%), and selected TnAK mutants complemented E. coli growth more strongly than homologous BsAK variants. Sequencing of functional clones from each library also identified a greater dispersion of fragmentation sites within TnAK. Nondisruptive fission sites were observed within the AMP binding and core domains of both AK homologs. However, only TnAK contained sites within the lid domain, which undergoes dynamic fluctuations that are critical for catalysis. These findings implicate the flexible lid domain as having an increased sensitivity to fission events at physiological temperatures. In addition, they provide evidence that comparisons of nondisruptive fission sites in homologous proteins could be useful for finding dynamic regions whose conformational fluctuations are important for function, and they show that the discovery of protein fragments that cooperatively function in mesophiles can be aided by the use of thermophilic enzymes as starting points for protein design.

Mesophilic and hyperthermophilic adenylate kinases differ in their tolerance to random fragmentation.

Adeno-associated virus (AAV)-derived vectors are promising gene delivery systems, and a number of design strategies have been pursued to improve their performance. For example, genetic insertion of proteins into the capsid may be used to achieve vector retargeting, reduced immunogenicity, or to track vector transport. Unfortunately, rational approaches to genetic insertion have experienced limited success due to the unpredictable context-dependent nature of protein folding and the complexity of the capsid's macroassembly. We report the construction and use of a frame-enriched DNase-based random insertion library based on AAV2 cap, called pAAV2_RaPID (Random Peptide Insertion by DNase). The fluorescent mCherry protein was inserted randomly throughout the AAV2 capsid and the library was selected for fluorescent and infectious variants. A capsid site was identified in VP3 that can tolerate the large protein insertion. In contrast to previous efforts to incorporate fluorescent proteins into the AAV2 capsid, the isolated mCherry mutant maintains native infectivity while displaying robust fluorescence. Collectively, these results demonstrate that the pAAV2_RaPID platform library can be used to create fully infectious AAV vectors carrying large functional protein domains on the capsid.Molecular Therapy - Nucleic Acids (2012) 1, e54; doi:10.1038/mtna.2012.46; published online 13 November 2012.

Random Insertion of mCherry Into VP3 Domain of Adeno-associated Virus Yields Fluorescent Capsids With no Loss of Infectivity.

We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus-receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.

Tunable protease-activatable virus nanonodes.

Gene delivery vectors that are activated by external stimuli may allow improved control over the location and the degree of gene expression in target populations of cells. Light is an attractive stimulus because it does not cross-react with cellular signaling networks, has negligible toxicity, is noninvasive, and can be applied in space and time with unparalleled precision. We used the previously engineered red (R)/far-red (FR) light-switchable protein phytochrome B (PhyB) and its R light dependent interaction partner phytochrome interacting factor 6 (PIF6) from Arabidopsis thaliana to engineer an adeno-associated virus (AAV) platform whose gene delivery efficiency is controlled by light. Upon exposure to R light, AAV engineered to display PIF6 motifs on the capsid bind to PhyB tagged with a nuclear localization sequence (NLS), resulting in significantly increased translocation of viruses into the host cell nucleus and overall gene delivery efficiency. By modulating the ratio of R to FR light, the gene delivery efficiency can be tuned to as little as 35% or over 600% of the unengineered AAV. We also demonstrate spatial control of gene delivery using projected patterns of codelivered R and FR light. Overall, our successful use of light-switchable proteins in virus capsid engineering extends these important optogenetic tools into the adjacent realm of nucleic acid delivery and enables enhanced, tunable, and spatially controllable regulation of viral gene delivery. Our current light-triggered viral gene delivery prototype may be broadly useful for genetic manipulation of cells ex vivo or in vivo in transgenic model organisms, with the ultimate prospect of achieving dose- and site-specific gene expression profiles for either therapeutic (e.g., regenerative medicine) or fundamental discovery research efforts.

Light-Activated Nuclear Translocation of Adeno-Associated Virus Nanoparticles Using Phytochrome B for Enhanced, Tunable, and Spatially Programmable Gene Delivery.

Article	Year
Effects of TRPA1 agonists mustard oil and cinnamaldehyde on lumbar spinal wide-dynamic range neuronal responses to innocuous and noxious cutaneous stimuli in rats. Journal of neurophysiology\| PubMed ID: 17942619	2008
Mesophilic and hyperthermophilic adenylate kinases differ in their tolerance to random fragmentation. Journal of molecular biology\| PubMed ID: 21145325	2011
Random Insertion of mCherry Into VP3 Domain of Adeno-associated Virus Yields Fluorescent Capsids With no Loss of Infectivity. Molecular therapy. Nucleic acids\| PubMed ID: 23629029	2012
Tunable protease-activatable virus nanonodes. ACS nano\| PubMed ID: 24796495	2014
Light-Activated Nuclear Translocation of Adeno-Associated Virus Nanoparticles Using Phytochrome B for Enhanced, Tunable, and Spatially Programmable Gene Delivery. ACS nano\| PubMed ID: 26618393	2016

Justin Judd

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