Tool Developed To Silence Genes In Specific Tissues Using RNAi

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Tool Developed To Silence Genes In Specific Tissues Using RNAi

http://www.medicalnewstoday.com/medicalnews.php?newsid=36213

Researchers at The University of Texas M. D. Cancer Center

say they have jumped a significant hurdle in the use of RNA

interference (RNAi), believed by many to be the ultimate tool to both

decode the function of individual genes in the human genome and to

treat disease.

Reporting in the journal Genes and Development, investigators have

developed a simple way to use the RNAi approach to silence a selected

gene in a specific tissue in a mouse to determine the function of

that targeted gene.

Previously reported approaches to achieve this were either

technically cumbersome, not generally applicable, or only achieved

transient knockdown of the target gene.

" Having a tool that will allow us to knockdown the expression of any

given gene in any specific tissue or cell type represents a

significant advance in the field, " says the study's lead

investigator, Miles Wilkinson, Ph.D., a professor in the Department

of Immunology.

For example, this method could potentially be used in humans " to

knockdown the expression of mutant or overexpressed genes that cause

human diseases, including cancer, " Wilkinson says. " Scientists and

clinicians can use it to reduce the expression of the target gene in

a single or limited numbers of cell types or tissues, thereby

reducing side effects. "

Equally, the technique can help researchers determine the function of

a single gene in a single tissue, which " is potentially a powerful

investigative tool, " Wilkinson says. " By silencing a particular gene

in a specific tissue, you can learn what the function of that gene is

in that particular tissue without blocking its essential functions in

other tissues. "

In their study, Wilkinson and his research team demonstrated how well

their tool worked by silencing the WT1 tumor suppressor gene in the

testes of mice. They found this gene is important in the production

of healthy sperm by encoding a regulatory protein called a

transcription factor that controls the formation of adherens

junctions, or the cell-to-cell contacts between nurse cells and the

germ cells that ultimately become sperm.

Using RNAi to silence WT1, therefore, led to the discovery that WT1

is the first transcription factor shown to regulate the formations of

these junctions, Wilkinson says. " It is a transcription factor that

dictates both the formation of the testes in the embryo, and the

function of the testes after birth. "

Researchers worldwide have been trying to harness the power of RNAi

since it was discovered in 1998. RNAi rocked the world of science

because of the vast implications this natural cellular control system

potentially offered medicine.

RNAi is used by cells in many life forms to identify viral RNA, which

often enters a cell as a double strand. The organism's own RNA,

however, leaves the nucleus of cells (where it was produced by DNA)

as a single strand, to be decoded by ribosomes into the proteins that

perform all the work of the cell. RNAi, therefore, recognizes the

double strand of viral RNA as different, and sets in motion cell

machinery that destroys the invading RNA. This involves cutting up

the double-stranded RNA, separating it into single strands, and

destroying other single-stranded RNA molecules that are complementary

to those small RNA segments.

Scientists quickly realized that if they could produce a double-

stranded RNA that mimics the RNA produced by a gene they wish to

silence, RNAi would do the job for them. Producing the decoy " small

RNAs " that trigger RNAi has become a fairly simple process, Wilkinson

says, but the difficulty has been to use these matches only in

specific tissues, and to figure out a way to make this " treatment "

last. The work by the M. D. team has come up with a solution

that solves both problems.

The technique they developed involves use of two different " modules "

that can be swapped in and out of the backbone of a vector. One is a

small stem loop designed to complement the RNA produced by the gene

they wish to silence, and the other is a " promoter " that provides

expression specific to the tissue they want to target. " There are

whole batteries of different promoters, ones specifically for skin,

or different parts of the brain, or whatever organ or tissue you are

likely to want, " Wilkinson says. " By swapping out either of these

modules, you have the potential to silence any gene in any tissue you

might want, " he said.

In their study, the researchers made a vector with a promoter

specific for nurse cells in the testis, so that the small RNA that is

associated with WT1 is only made in this cell type, thereby reducing

WT1 levels that are only in the nurse cells. Other organs, like

kidney, that need WT1 to function are not affected, Wilkinson says.

" We are very excited about the potential of this approach, and hope

that is solves what had become a significant roadblock to effective

use of RNAi, " he says.

The study was funded by the National Institutes of Health. The co-

authors from M. D. Cancer Center are Manjeet Rao, Ph.D.,

Pham, J. Saadi Iman, MacLean, Ph.D., Deepa Murali, Ph.D.,

and Yashudi Furuta, Ph.D., Amiya Sinha-Hilim, Ph.D., from the UCLA

School of Medicine also contributed.