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.