12 research outputs found
Delivery of CRISPR-Cas9 into Mouse Zygotes by Electroporation.
The CRISPR-Cas9 system in bacteria and archaea has recently been exploited for genome editing in various model organisms, including the mice. In this scheme, components of the CRISPR-Cas9 system are delivered into the mouse zygote and mutant mice carrying genetic modifications derived. Although microinjection has been the technology of choice, electroporation has also emerged and been proven to be effective delivering CRISPR-Cas9 reagents into the mouse zygote. Here, we describe the experimental protocol employing electroporation to deliver CRISPR-Cas9 reagents into mouse embryos and derive gene-edited mutant mice
Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells
The RNA interference-mediated gene silencing approach is promising for therapies based on the targeted inhibition of disease-relevant genes. Electropermeabilization is one of the nonviral methods successfully used to transfer siRNA into living cells in vitro and in vivo. Although this approach is effective in the field of gene silencing by RNA interference, very little is known about the basic processes supporting siRNA transfer. In this study, we investigated, by direct visualization at the single-cell level, the delivery of Alexa Fluor 546-labeled siRNA into murine melanoma cells stably expressing the enhanced green fluorescent protein (EGFP) as a target gene. The electrotransfer of siRNA was quantified by time lapse fluorescence microscopy and was correlated with the silencing of egfp expression. A direct transfer into the cell cytoplasm of the negatively charged siRNA was observed across the plasma membrane exclusively on the side facing the cathode. When added after electropulsation, the siRNA was inefficient for gene silencing because it did not penetrate the cells. Therefore, we report that an electric field acts on both the permeabilization of the cell plasma membrane and on the electrophoretic drag of the negatively charged siRNA molecules from the bulk phase into the cytoplasm. The transfer kinetics of siRNA are compatible with the creation of nanopores, which are described with the technique of synthetic nanopores. The mechanism involved was clearly specific for the physico-chemical properties of the electrotransferred molecule and was different from that observed with small molecules or plasmid DNA
High-yield nontoxic gene transfer through conjugation of the CM\u2081\u2088-Tat\u2081\u2081 chimeric peptide with nanosecond electric pulses
We report a novel nontoxic, high-yield, gene delivery system based on the synergistic use of nanosecond electric pulses (NPs) and nanomolar doses of the recently introduced CM18-Tat11 chimeric peptide (sequence of KWKLFKKIGAVLKVLTTGYGRKKRRQRRR, residues 1-7 of cecropin-A, 2-12 of melittin, and 47-57 of HIV-1 Tat protein). This combined use makes it possible to drastically reduce the required CM18-Tat11 concentration and confines stable nanopore formation to vesicle membranes followed by DNA release, while no detectable perturbation of the plasma membrane is observed. Two different experimental assays are exploited to quantitatively evaluate the details of NPs and CM18-Tat11 cooperation: (i) cytofluorimetric analysis of the integrity of synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles exposed to CM18-Tat11 and NPs and (ii) the in vitro transfection efficiency of a green fluorescent protein-encoding plasmid conjugated to CM18-Tat11 in the presence of NPs. Data support a model in which NPs induce membrane perturbation in the form of transient pores on all cellular membranes, while the peptide stabilizes membrane defects selectively within endosomes. Interestingly, atomistic molecular dynamics simulations show that the latter activity can be specifically attributed to the CM18 module, while Tat11 remains essential for cargo binding and vector subcellular localization. We argue that this result represents a paradigmatic example that can open the way to other targeted delivery protocols
A StructureâActivity Relationship Study of Bimodal BODIPYâLabeled PSMAâTargeting Bioconjugates
The aim of this study was to identify a highâaffinity BODIPY peptidomimetic that targets the prostateâspecific membrane antigen (PSMA) as a potential bimodal imaging probe for prostate cancer. For the structureâactivity study, several BODIPY (difluoroboron dipyrromethene) derivatives with varying spacers between the BODIPY dye and the PSMA GluâCOâLys binding motif were prepared. Corresponding affinities were determined by competitive binding assays in PSMAâpositive LNCaP cells. One compound was identified with comparable affinity (IC(50)=21.5±0.1 nM) to GluâCOâLysâAhxâHBEDâCC (PSMAâ11) (IC(50)=18.4±0.2 nM). Radiolabeling was achieved by Lewisâacidâmediated (19)F/(18)F exchange in moderate molar activities (âŒ0.7â
MBqânmol(â1)) and high radiochemical purities (>99â%) with mean radiochemical yields of 20â30â%. Cell internalization of the (18)Fâlabeled highâaffinity conjugate was demonstrated in LNCaP cells showing gradual increasing PSMAâmediated internalization over time. By fluorescence microscopy, localization of the highâaffinity BODIPYâPSMA conjugate was found in the cell membrane at early time points and also in subcellular compartments at later time points. In summary, a highâaffinity BODIPYâPSMA conjugate has been identified as a suitable candidate for the development of PSMAâspecific dualâimaging agents