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Interfering RNA silences genes in 'slippery' immune cells

o 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
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Source:Children's Hospital of Philadelphia


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