Limits...
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

Variation of percentage of hatched embryos labeled with different doses of antisense reagents. Rhodamine-dextran (RhDextran) or its conjugates of antisense 2'-O-methyl oligoribonucleotides against let-7, lin-4, lsy-6, mir-237 were injected into gonads of N2 worms at three different concentrations. Each time we collected 50 or more labelled embryos and counted hatched larvae the next day. Error bars are standard errors of two independent injections. Examination of embryos that failed to hatch revealed that most of them were arrested around 50-cell stage. Embryos, once hatched, developed into adults that appeared to be grossly normal.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2864223&req=5

Figure 10: Variation of percentage of hatched embryos labeled with different doses of antisense reagents. Rhodamine-dextran (RhDextran) or its conjugates of antisense 2'-O-methyl oligoribonucleotides against let-7, lin-4, lsy-6, mir-237 were injected into gonads of N2 worms at three different concentrations. Each time we collected 50 or more labelled embryos and counted hatched larvae the next day. Error bars are standard errors of two independent injections. Examination of embryos that failed to hatch revealed that most of them were arrested around 50-cell stage. Embryos, once hatched, developed into adults that appeared to be grossly normal.

Mentions: Among four miRNAs tested (lin-4, lsy-6, let-7 and mir-42), the dose of antisense reagents required for the effective inhibition of individual miRNA varied from as low as three micromolar (lsy-6 and mir-42) to as high as tens of micromolar (let-7). Two factors may account for the difference in the apparent potency of these antisense reagents. First, the cellular expression level of different miRNAs varies over a wide range [23], so a higher concentration of antisense reagents is needed to block more abundant miRNAs. Second, to inhibit a miRNA that is expressed late in larval development, it would require a higher concentration of reagent in the zygote to compensate for the dilution effect of cell division and larval growth. let-7, for example, is not expressed until the third larval stage and functions in the fourth stage [19]. By that time, the antisense reagent would be further diluted as animals grow and expand in size. In principle, a higher concentration of antisense reagents can be used to overcome the dilution effect. However, it should be cautioned that, as we raised the dose, we also noticed that an increasing number of embryos failed to hatch at concentrations above 50 μM. The extent to which these antisense reagents affect embryo development seemed to vary. Among the reagents tested, antisense reagents (antimirs) against lin-4 and mir-237 were best tolerated, with nearly 100% of the embryos hatched normally at 100 μM. By comparison, the antimir against let-7 was least tolerated, with close to 40% of embryos hatched at 100 μM (Figure 10). We have not yet explored the causes for this differential effect, although we anticipate that the future development of other bioconjugates employing different classes of oligonucleotides, for example locked nucleic acids or 2'-O-methoxyethyl oligoribonucleotides, may offer an improvement on the potency with concomitant reduction of the perturbation on embryo development.


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

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

Variation of percentage of hatched embryos labeled with different doses of antisense reagents. Rhodamine-dextran (RhDextran) or its conjugates of antisense 2'-O-methyl oligoribonucleotides against let-7, lin-4, lsy-6, mir-237 were injected into gonads of N2 worms at three different concentrations. Each time we collected 50 or more labelled embryos and counted hatched larvae the next day. Error bars are standard errors of two independent injections. Examination of embryos that failed to hatch revealed that most of them were arrested around 50-cell stage. Embryos, once hatched, developed into adults that appeared to be grossly normal.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: Variation of percentage of hatched embryos labeled with different doses of antisense reagents. Rhodamine-dextran (RhDextran) or its conjugates of antisense 2'-O-methyl oligoribonucleotides against let-7, lin-4, lsy-6, mir-237 were injected into gonads of N2 worms at three different concentrations. Each time we collected 50 or more labelled embryos and counted hatched larvae the next day. Error bars are standard errors of two independent injections. Examination of embryos that failed to hatch revealed that most of them were arrested around 50-cell stage. Embryos, once hatched, developed into adults that appeared to be grossly normal.
Mentions: Among four miRNAs tested (lin-4, lsy-6, let-7 and mir-42), the dose of antisense reagents required for the effective inhibition of individual miRNA varied from as low as three micromolar (lsy-6 and mir-42) to as high as tens of micromolar (let-7). Two factors may account for the difference in the apparent potency of these antisense reagents. First, the cellular expression level of different miRNAs varies over a wide range [23], so a higher concentration of antisense reagents is needed to block more abundant miRNAs. Second, to inhibit a miRNA that is expressed late in larval development, it would require a higher concentration of reagent in the zygote to compensate for the dilution effect of cell division and larval growth. let-7, for example, is not expressed until the third larval stage and functions in the fourth stage [19]. By that time, the antisense reagent would be further diluted as animals grow and expand in size. In principle, a higher concentration of antisense reagents can be used to overcome the dilution effect. However, it should be cautioned that, as we raised the dose, we also noticed that an increasing number of embryos failed to hatch at concentrations above 50 μM. The extent to which these antisense reagents affect embryo development seemed to vary. Among the reagents tested, antisense reagents (antimirs) against lin-4 and mir-237 were best tolerated, with nearly 100% of the embryos hatched normally at 100 μM. By comparison, the antimir against let-7 was least tolerated, with close to 40% of embryos hatched at 100 μM (Figure 10). We have not yet explored the causes for this differential effect, although we anticipate that the future development of other bioconjugates employing different classes of oligonucleotides, for example locked nucleic acids or 2'-O-methoxyethyl oligoribonucleotides, may offer an improvement on the potency with concomitant reduction of the perturbation on embryo development.

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