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Interfering RNA Silences Genes In 'Slippery' Immune Cells -- Novel Lab Technique Expands Researchers' Toolbox
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.
About The Children's Hospital of Philadelphia: The Children's Hospital
of Philadelphia was founded in 1855 as the nation's first pediatric
hospital. Through its long-standing commitment to providing exceptional
patient care, training new generations of pediatric healthcare
professionals and pioneering major research initiatives, Children's
Hospital has fostered many discoveries that have benefited children
worldwide. Its pediatric research program is among the largest in the
country, ranking second in National Institutes of Health funding. In
addition, its unique family-centered care and public service programs have
brought the 430-bed hospital recognition as a leading advocate for children
and adolescents. For more information, visit http://www.chop.edu.
The Children's Hospital of Philadelphia
http://www.chop.edu
Interferenþa ARN tãcerile Genele În "Slippery" celulele imune - Novel Lab Tehnica extinde cercetãtorilor Toolbox - Interfering RNA Silences Genes In 'Slippery' Immune Cells -- Novel Lab Technique Expands Researchers' Toolbox - articole medicale engleza - startsanatate