A technical advance in laboratory techniques may provide biology researchers broader access to RNA interference, a process of blocking the activity of targeted genes. RNA interference has recently emerged as an important tool in studying how genes function in normal biological processes and in disease.
Writing in the Journal of Immunological Methods, published online on March 24, a research team from The Children’s Hospital of Philadelphia combined laboratory technologies in using RNA interference to manipulate human T cells. T cells are immune cells that circulate in the blood, with important roles in autoimmune diseases, infectious diseases and some cancers.
"T cells have previously been difficult to modify with interfering RNA, being more mobile than other cell types that typically remain stationary in cell cultures," said study leader Terri H. Finkel, M.D., Ph.D., chief of Rheumatology at The Children’s Hospital of Philadelphia. "Our approach achieves results comparable to the conventional technique, which uses synthetic small interfering RNA but is very expensive and in short supply. We expect our technique to expand the toolbox for scientists doing research in immunology."
RNA interference (RNAi), which naturally occurs in cells, is a process in which brief RNA sequences, called small interfering RNA (siRNA) block signals from a particular gene. This process, called gene silencing, inhibits the gene from carrying out its function of creating a protein or another gene product. The body often uses RNAi as a defense against the action of hostile viruses.
Over the past few years, biomedical researchers have been investigating how they might eventually harness RNAi in new medicines. Another line of research uses RNAi as a research tool, investigating the functions of specific genes by studying what happens when RNAi temporarily silences them--a process calling "knocking down" the gene.
The research by Dr. Finkel’s team aims to extend RNAi to a wider pool of researchers by making the technique less expensive and more widely available, as well as adapting it to T cells, a cell type previously intractable to such manipulation. Their technique combines three technologies already accessible to lab investigators: nucleofection, siRNA expression cassettes, and siRNA expression vectors. Nucleofection technology uses specialized solutions and electrical pulses to temporarily open a cell nucleus. Into the nucleus, researchers insert a payload of DNA.
The DNA holds a sequence of genetic code that produces a specific siRNA after it enters a nucleus. The researchers encased the DNA within an siRNA expression cassette (SEC), an inexpensive, quickly synthesized product that carries genetic sequences to regulate the gene activity that yields an siRNA. After the researchers tested a variety of SECs to determine which is the most effective, they inserted the desired SEC into a vector, a biological agent that inserts itself into a target cell’s nucleus more efficiently than an unaccompanied cassette.
The researchers first tested their approach by introducing a gene for green fluorescent protein into human T cells, and using siRNA to inhibit that gene’s expression, and dim its fluorescent glow.
They then applied their approach to HALP, a gene naturally active in T cells. Dr. Finkel previously discovered and named HALP, an acronym for "HIV-associated life preserver," showing that it had a role in prolonging HIV infection by helping HIV-infected T cells survive attack by the immune system.
Using siRNA and their laboratory techniques, the investigators succeeded in "knocking down," that is, decreasing gene expression by HALP. Because their previous research strongly suggests that HALP promotes latent HIV infection, the new technique has a potential application to HIV treatment. "The siRNA may represent a suicide vector: by knocking down HALP it may allow HIV-infected cells to self-destruct, thus eliminating a hiding place for the virus," said Dr. Finkel.
"More broadly," she added, "the technique could theoretically be directed against other immune-related diseases, by silencing harmful genes active in T cells."
Dr. Finkel’s co-authors, all from The Children’s Hospital of Philadelphia, were Jiyi Yin, Ph.D., Zhengyu Ma, Nithianandan Selliah, Ph.D., Debra K. Shivers and Randy Q. Cron, M.D., Ph.D. National Institutes of Health grants supported the research, along with support from the University of Pennsylvania Center for AIDS Research and the University’s Cancer Center, the Bender Foundation, the Joseph Lee Hollander Chair at The Children’s Hospital of Philadelphia, and the W. W. Smith Charitable Trust.
"Effective Gene Suppression Using Small Interfering RNA in Hard-to-Transfect Human T Cells." Journal of Immunological Methods. In press, published online March 24, 2006.
John Ascenzi | EurekAlert!
Study shines light on brain cells that coordinate movement
26.06.2017 | University of Washington Health Sciences/UW Medicine
New insight into a central biological dogma on ion transport
26.06.2017 | Aarhus University
An international team of scientists has proposed a new multi-disciplinary approach in which an array of new technologies will allow us to map biodiversity and the risks that wildlife is facing at the scale of whole landscapes. The findings are published in Nature Ecology and Evolution. This international research is led by the Kunming Institute of Zoology from China, University of East Anglia, University of Leicester and the Leibniz Institute for Zoo and Wildlife Research.
Using a combination of satellite and ground data, the team proposes that it is now possible to map biodiversity with an accuracy that has not been previously...
Heatwaves in the Arctic, longer periods of vegetation in Europe, severe floods in West Africa – starting in 2021, scientists want to explore the emissions of the greenhouse gas methane with the German-French satellite MERLIN. This is made possible by a new robust laser system of the Fraunhofer Institute for Laser Technology ILT in Aachen, which achieves unprecedented measurement accuracy.
Methane is primarily the result of the decomposition of organic matter. The gas has a 25 times greater warming potential than carbon dioxide, but is not as...
Hydrogen is regarded as the energy source of the future: It is produced with solar power and can be used to generate heat and electricity in fuel cells. Empa researchers have now succeeded in decoding the movement of hydrogen ions in crystals – a key step towards more efficient energy conversion in the hydrogen industry of tomorrow.
As charge carriers, electrons and ions play the leading role in electrochemical energy storage devices and converters such as batteries and fuel cells. Proton...
Scientists from the Excellence Cluster Universe at the Ludwig-Maximilians-Universität Munich have establised "Cosmowebportal", a unique data centre for cosmological simulations located at the Leibniz Supercomputing Centre (LRZ) of the Bavarian Academy of Sciences. The complete results of a series of large hydrodynamical cosmological simulations are available, with data volumes typically exceeding several hundred terabytes. Scientists worldwide can interactively explore these complex simulations via a web interface and directly access the results.
With current telescopes, scientists can observe our Universe’s galaxies and galaxy clusters and their distribution along an invisible cosmic web. From the...
Temperature measurements possible even on the smallest scale / Molecular ruby for use in material sciences, biology, and medicine
Chemists at Johannes Gutenberg University Mainz (JGU) in cooperation with researchers of the German Federal Institute for Materials Research and Testing (BAM)...
19.06.2017 | Event News
13.06.2017 | Event News
13.06.2017 | Event News
26.06.2017 | Life Sciences
26.06.2017 | Physics and Astronomy
26.06.2017 | Information Technology