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Inhibiting miRNA in Caenorhabditis elegans using a potent and selective antisense reagent.

Zheng G, Ambros V, Li WH - Silence (2010)

Bottom Line: These reagents were synthesized by conjugating dextran with 2'-O-methyl oligoribonucleotide.We show that these reagents can be used combinatorially to inhibit more than one miRNA in the same animal.This class of antisense reagents represents a new addition to the toolkit for studying miRNA in C. elegans.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Cell Biology and of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9039, USA. wen-hong.li@UTSouthwestern.edu.

ABSTRACT

Background: Antisense reagents can serve as efficient and versatile tools for studying gene function by inhibiting nucleic acids in vivo. Antisense reagents have particular utility for the experimental manipulation of the activity of microRNAs (miRNAs), which are involved in the regulation of diverse developmental and physiological pathways in animals. Even in traditional genetic systems, such as the nematode Caenorhabditis elegans, antisense reagents can provide experimental strategies complementary to mutational approaches. Presently no antisense reagents are available for inhibiting miRNAs in the nematode C. elegans.

Results: We have developed a new class of fluorescently labelled antisense reagents to inhibit miRNAs in developing worms. These reagents were synthesized by conjugating dextran with 2'-O-methyl oligoribonucleotide. The dextran-conjugated antisense reagents can be conveniently introduced into the germline of adult hermaphrodites and are transmitted to their progeny, where they efficiently and specifically inhibit a targeted miRNA in different tissues, including the hypodermis, the vulva and the nervous system. We show that these reagents can be used combinatorially to inhibit more than one miRNA in the same animal.

Conclusion: This class of antisense reagents represents a new addition to the toolkit for studying miRNA in C. elegans. Combined with numerous mutants or reporter stains available, these reagents should provide a convenient approach to examine genetic interactions that involve miRNA, and may facilitate studying functions of miRNAs, especially ones whose deletion strains are difficult to generate.See related research article: http://jbiol.com/content/9/3/20.

No MeSH data available.


Related in: MedlinePlus

Inhibit let-7 with Rhdextran-(as-2'OMelet-7)1. (a) Dose response of dextran-(as-2'OMelet-7)1 in causing bursting vulva or no alae formation. Error bars are standrad errors of two independent injections. After each injection, 50 or more embryos from injected worms were randomly picked and scored for bursting vulva or absense of alae when they reached adults. (b) An example image showing the bursting vulva phenotype.
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Figure 8: Inhibit let-7 with Rhdextran-(as-2'OMelet-7)1. (a) Dose response of dextran-(as-2'OMelet-7)1 in causing bursting vulva or no alae formation. Error bars are standrad errors of two independent injections. After each injection, 50 or more embryos from injected worms were randomly picked and scored for bursting vulva or absense of alae when they reached adults. (b) An example image showing the bursting vulva phenotype.

Mentions: Another miRNA, let-7, controls the larval-to-adult transition by repressing the translation of lin-41 and hbl-1. Mutations in let-7 lead to a retarded terminal differentiation of seam cells, which results in the elimination of alae and a bursting of the animal at the vulva as the animal undergoes the fourth molt [19]. We observed a dose-dependent bursting or no-alae phenotype, consistent with inhibition of let-7 activity in the progeny of hermaphrodites injected with Rhdextran-(as-2'OMelet-7)1 (Figure 8). When Rhdextran-(as-2'OMelet-7)1 was injected at 20 μM, it caused bursting vulva in about 40% of labelled worms and eliminated the formation of alae in nearly 80% of worms. Raising the concentration of Rhdextran-(as-2'OMelet-7)1 to 50 μM or above increased the penetrance, and none of the labelled animals showed alae when they reached young adults. In contrast, an injection of Rhdextran-(as-2'OMemir-84)1 directed against another let-7 family microRNA caused no observable phenotype. Since mir-84 mutations do not cause visible phenotypes [20], this result is consistent with these antisense reagents inhibiting the targeted miRNA with high specificity. Further, in a lin-41 non mutant, lin-41(ma104), Rhdextran-(as-2'OMelet-7)1 (50 μM) failed to produce the bursting vulva or alae defect in labelled worms (n = 12). This again suggested that these antisense reagents acted specifically by blocking their corresponding microRNAs.


Inhibiting miRNA in Caenorhabditis elegans using a potent and selective antisense reagent.

Zheng G, Ambros V, Li WH - Silence (2010)

Inhibit let-7 with Rhdextran-(as-2'OMelet-7)1. (a) Dose response of dextran-(as-2'OMelet-7)1 in causing bursting vulva or no alae formation. Error bars are standrad errors of two independent injections. After each injection, 50 or more embryos from injected worms were randomly picked and scored for bursting vulva or absense of alae when they reached adults. (b) An example image showing the bursting vulva phenotype.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2864223&req=5

Figure 8: Inhibit let-7 with Rhdextran-(as-2'OMelet-7)1. (a) Dose response of dextran-(as-2'OMelet-7)1 in causing bursting vulva or no alae formation. Error bars are standrad errors of two independent injections. After each injection, 50 or more embryos from injected worms were randomly picked and scored for bursting vulva or absense of alae when they reached adults. (b) An example image showing the bursting vulva phenotype.
Mentions: Another miRNA, let-7, controls the larval-to-adult transition by repressing the translation of lin-41 and hbl-1. Mutations in let-7 lead to a retarded terminal differentiation of seam cells, which results in the elimination of alae and a bursting of the animal at the vulva as the animal undergoes the fourth molt [19]. We observed a dose-dependent bursting or no-alae phenotype, consistent with inhibition of let-7 activity in the progeny of hermaphrodites injected with Rhdextran-(as-2'OMelet-7)1 (Figure 8). When Rhdextran-(as-2'OMelet-7)1 was injected at 20 μM, it caused bursting vulva in about 40% of labelled worms and eliminated the formation of alae in nearly 80% of worms. Raising the concentration of Rhdextran-(as-2'OMelet-7)1 to 50 μM or above increased the penetrance, and none of the labelled animals showed alae when they reached young adults. In contrast, an injection of Rhdextran-(as-2'OMemir-84)1 directed against another let-7 family microRNA caused no observable phenotype. Since mir-84 mutations do not cause visible phenotypes [20], this result is consistent with these antisense reagents inhibiting the targeted miRNA with high specificity. Further, in a lin-41 non mutant, lin-41(ma104), Rhdextran-(as-2'OMelet-7)1 (50 μM) failed to produce the bursting vulva or alae defect in labelled worms (n = 12). This again suggested that these antisense reagents acted specifically by blocking their corresponding microRNAs.

Bottom Line: These reagents were synthesized by conjugating dextran with 2'-O-methyl oligoribonucleotide.We show that these reagents can be used combinatorially to inhibit more than one miRNA in the same animal.This class of antisense reagents represents a new addition to the toolkit for studying miRNA in C. elegans.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Cell Biology and of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9039, USA. wen-hong.li@UTSouthwestern.edu.

ABSTRACT

Background: Antisense reagents can serve as efficient and versatile tools for studying gene function by inhibiting nucleic acids in vivo. Antisense reagents have particular utility for the experimental manipulation of the activity of microRNAs (miRNAs), which are involved in the regulation of diverse developmental and physiological pathways in animals. Even in traditional genetic systems, such as the nematode Caenorhabditis elegans, antisense reagents can provide experimental strategies complementary to mutational approaches. Presently no antisense reagents are available for inhibiting miRNAs in the nematode C. elegans.

Results: We have developed a new class of fluorescently labelled antisense reagents to inhibit miRNAs in developing worms. These reagents were synthesized by conjugating dextran with 2'-O-methyl oligoribonucleotide. The dextran-conjugated antisense reagents can be conveniently introduced into the germline of adult hermaphrodites and are transmitted to their progeny, where they efficiently and specifically inhibit a targeted miRNA in different tissues, including the hypodermis, the vulva and the nervous system. We show that these reagents can be used combinatorially to inhibit more than one miRNA in the same animal.

Conclusion: This class of antisense reagents represents a new addition to the toolkit for studying miRNA in C. elegans. Combined with numerous mutants or reporter stains available, these reagents should provide a convenient approach to examine genetic interactions that involve miRNA, and may facilitate studying functions of miRNAs, especially ones whose deletion strains are difficult to generate.See related research article: http://jbiol.com/content/9/3/20.

No MeSH data available.


Related in: MedlinePlus