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Viral delivery of small-hairpin RNAs for reducing gene expression in the rodent brain.

Lasek AW, Heberlein U - Alcohol Res Health (2008)

Bottom Line: This article reviews a strategy to ensure long-term expression of a type of interfering RNA molecule-small-hairpin RNAs (shRNAs)-through the use of viral delivery systems (i.e., vectors).Expression of these shRNAs leads to the destruction of the intermediary molecules (i.e., messenger RNA [mRNA] molecules) generated during the expression of the target gene.

View Article: PubMed Central - PubMed

Affiliation: Ernest Gallo Clinic and Research Center at the University of California at San Francisco, Emeryville, California.

ABSTRACT
This article reviews a strategy to ensure long-term expression of a type of interfering RNA molecule-small-hairpin RNAs (shRNAs)-through the use of viral delivery systems (i.e., vectors). Expression of these shRNAs leads to the destruction of the intermediary molecules (i.e., messenger RNA [mRNA] molecules) generated during the expression of the target gene.

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Example of the effectiveness of knock-down of expression of the μ-opioid receptor (MOR) by different small-hairpin RNAs (shRNAs). Shown is the relative level of MOR expression after introduction of three different shRNAs or a control construct (i.e., a construct containing a “scrambled” shRNA that should not target any mouse or rat gene) into cells stably expressing MOR. The results show that at 48 hours after the introduction of the three constructs and the control, construct MOR2 is most effective, essentially shutting down the cell’s MOR production.
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f12-arh-31-3-259: Example of the effectiveness of knock-down of expression of the μ-opioid receptor (MOR) by different small-hairpin RNAs (shRNAs). Shown is the relative level of MOR expression after introduction of three different shRNAs or a control construct (i.e., a construct containing a “scrambled” shRNA that should not target any mouse or rat gene) into cells stably expressing MOR. The results show that at 48 hours after the introduction of the three constructs and the control, construct MOR2 is most effective, essentially shutting down the cell’s MOR production.

Mentions: An essential step in using this approach to reduce or eliminate expression of a specific gene is the design of suitable shRNAs. In general, shRNAs are designed based on a variety of criteria developed by Reynolds and colleagues (2004). To simplify the process, researchers can use publicly accessible Web sites to design shRNAs for their specific experiments, such as the Thermo Scientific Dharmacon siDesign Center (available at: http://www.dharmacon.com/DesignCenter/DesignCenterPage.aspx) or the siRNA Web site at the Whitehead Institute (available at: http://jura.wi.mit.edu/bioc/siRNAext/). For each gene, three candidate target sequences of 19 nucleotides are chosen. Then, nucleotide chains (i.e., oligonucleotides) are synthesized in the laboratory that contain complementary 19-nucleotide target sequences with the loop sequence inserted between the two complementary sequences (so that they can form the characteristic stem-loop structure of the shRNA) and a signal sequence that stops expression of the shRNA at the right site. Once these oligonucleotides have been introduced into the lentivirus vector, they are first tested in cultured cells to determine if and how effectively they can knock down target gene expression (see figure 12).


Viral delivery of small-hairpin RNAs for reducing gene expression in the rodent brain.

Lasek AW, Heberlein U - Alcohol Res Health (2008)

Example of the effectiveness of knock-down of expression of the μ-opioid receptor (MOR) by different small-hairpin RNAs (shRNAs). Shown is the relative level of MOR expression after introduction of three different shRNAs or a control construct (i.e., a construct containing a “scrambled” shRNA that should not target any mouse or rat gene) into cells stably expressing MOR. The results show that at 48 hours after the introduction of the three constructs and the control, construct MOR2 is most effective, essentially shutting down the cell’s MOR production.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3860491&req=5

f12-arh-31-3-259: Example of the effectiveness of knock-down of expression of the μ-opioid receptor (MOR) by different small-hairpin RNAs (shRNAs). Shown is the relative level of MOR expression after introduction of three different shRNAs or a control construct (i.e., a construct containing a “scrambled” shRNA that should not target any mouse or rat gene) into cells stably expressing MOR. The results show that at 48 hours after the introduction of the three constructs and the control, construct MOR2 is most effective, essentially shutting down the cell’s MOR production.
Mentions: An essential step in using this approach to reduce or eliminate expression of a specific gene is the design of suitable shRNAs. In general, shRNAs are designed based on a variety of criteria developed by Reynolds and colleagues (2004). To simplify the process, researchers can use publicly accessible Web sites to design shRNAs for their specific experiments, such as the Thermo Scientific Dharmacon siDesign Center (available at: http://www.dharmacon.com/DesignCenter/DesignCenterPage.aspx) or the siRNA Web site at the Whitehead Institute (available at: http://jura.wi.mit.edu/bioc/siRNAext/). For each gene, three candidate target sequences of 19 nucleotides are chosen. Then, nucleotide chains (i.e., oligonucleotides) are synthesized in the laboratory that contain complementary 19-nucleotide target sequences with the loop sequence inserted between the two complementary sequences (so that they can form the characteristic stem-loop structure of the shRNA) and a signal sequence that stops expression of the shRNA at the right site. Once these oligonucleotides have been introduced into the lentivirus vector, they are first tested in cultured cells to determine if and how effectively they can knock down target gene expression (see figure 12).

Bottom Line: This article reviews a strategy to ensure long-term expression of a type of interfering RNA molecule-small-hairpin RNAs (shRNAs)-through the use of viral delivery systems (i.e., vectors).Expression of these shRNAs leads to the destruction of the intermediary molecules (i.e., messenger RNA [mRNA] molecules) generated during the expression of the target gene.

View Article: PubMed Central - PubMed

Affiliation: Ernest Gallo Clinic and Research Center at the University of California at San Francisco, Emeryville, California.

ABSTRACT
This article reviews a strategy to ensure long-term expression of a type of interfering RNA molecule-small-hairpin RNAs (shRNAs)-through the use of viral delivery systems (i.e., vectors). Expression of these shRNAs leads to the destruction of the intermediary molecules (i.e., messenger RNA [mRNA] molecules) generated during the expression of the target gene.

Show MeSH