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Tools & Techniques: siRNA Delivery into Stem Cells, shRNA Expression Vector Construction, and More


A team of researchers from Rutgers University have developed a new approach to deliver siRNAs into neural stem cells.

While siRNAs have the potential to be an important tool in stem cell research, successful genetic manipulation of the cells requires that they maintain their viability for an extended period of time after transfection, the investigators wrote in Scientific Reports. “However, many of the conventional methods used to deliver siRNA into stem cells, including lipid-based transfections, cationic polyplexes, viral vectors, and electroporation techniques, result in significant cytotoxicity and undesirable side-effects.”

To address this issue, the team developed a nanotopography-mediated reverse uptake, or NanoRU, platform capable of delivering siRNAs into neural stem cells effectively and without toxicity.

According to the paper, NanoRU consists of a self-assembled silica nanoparticle monolayer coated with extracellular matrix proteins and the desired siRNA, and it was tested by delivering siRNAs against the transcription factor SOX9 into neural stem cells, enhancing neuronal differentiation and decreasing glial differentiation.

Even though they only examined siRNA delivery, the investigators wrote that they expect that the platform can be extended with modifications to a “wide range of nanomaterials and biomolecules,” including microRNAs and proteins.

Aiming to streamline the construction of shRNA expression vectors, researchers from Hubei University reported on a novel, single-step cloning method for the RNAi constructs that promises to be cheaper and quicker than existing approaches.

Traditionally, shRNA expression vectors for use in mammalian cells require the synthesis of two long and complementary oligos, which are annealed in test tubes and then inserted into the vectors, the team wrote in Analytical Biochemistry. Though straightforward, this process involves multiple steps, can be expensive, and opens the door to mutations during primer synthesis.

Some of these issues can be sidestepped by using a different approach based on overlap extension PCR, they noted. “However, mutations induced by DNA polymerase during either the initial extension step or the repeat cycling still cannot be avoided.”

The scientists previously discovered that an insert and a vector backbone that share homologous ends can form a recombinant vector in vivo when they are co-transformed into the same E. coli cell, and that the ends could be shortened to eight base pairs.

Based on these findings, they developed a mixing cloning strategy for lentiviral shRNA expression vectors that requires a “simple co-transformation of short oligonucleotides with vectors,” they wrote.

The vector can be created in a single step, and was used to successfully construct 30 lentiviral shRNAs expression vectors with no need for polymerase extension, digestion, and ligation.

Given the need for effective delivery strategies for RNAi-based cancer treatments, a group of researchers from the University of Utah have reported on the development of a new, non-viral delivery vehicle.

Naked siRNAs have largely proven ineffective in a therapeutic context due to rapid degradation by serum nucleases and low accumulation at target sites, the team wrote in Molecular Pharmaceutics. Additionally, uncoated siRNAs are large and negatively charged, hindering their cellular uptake.

In trying to tackle this hurdle, the researchers developed an siRNA carrier based on a combination of an arginine-grafted bioreducible polymer combined with an siRNA payload; microbubbles, which have been used for decades as ultrasound contrast agents; and ultrasound, which is capable of directing the microbubbles and rupturing them at target sites.

The researchers used the approach to deliver siRNAs against vascular endothelial growth factor in human ovarian cancer cells, significantly knocking down their target compared with naked siRNAs when incubated for a short time after sonication treatment.

Seeking to improve the accuracy of nanopores for nucleic acid detection, investigators from the University of Missouri have published details about a new polycationic probe that can simultaneously enrich and detect microRNAs in a nanopore.

Despite nanopores’ ability to detect nucleic acid biomarkers, non-specific interactions between the nanopore and other nucleic acid species in biofluid can contaminate the target signal and reduce accuracy — all of which limits their diagnostic potential, the team wrote in ACS Nano.

To solve this problem, the scientists devised a polycationic probe composed of a sequence of peptide nucleic acids specifically designed to capture a target miRNA, conjugated with a polycationic peptide lead.

“Upon hybridization, the positively charged peptide lead and the negatively charged miRNA together form a dipole,” they wrote. “This structure can be driven into the nanopore by a large electric field gradient around the nanopore opening.”

Any free nucleic acids without probe hybridization would carry a negative charge and migrate away from the pore opening. Thus, only the signatures for the miRNA probe complex and the probe alone in the nanopore are identified, eliminating interference signals from free nucleic acids.

The researchers tested the probes in vitro, but experiments in clinical samples have yet to be conducted.