Antisense reagents can serve as efficient and versatile tools for studying gene function by blocking nucleic acids in vivo. Presently no antisense reagents are available for inhibiting miRNAs in the nematode C. elegans. Such a technique would provide a convenient approach to study functions of miRNAs whose deletion strains are difficult to generate, to dissect functions of individual miRNAs that are clustered together, or to block intronic miRNAs without perturbing the function of the corresponding protein-coding genes.
Previous studies by Hutvagner et al. showed that an antisense 2′-O-methyl oligoribonucleotide injected into worm larvae could inhibit let-7 (Hutvagner et al., 2004) However, injection of worm larvae is technically very demanding, and this method cannot be applied to study miRNAs that are expressed early in development. We have developed a new class of fluorescently labeled antisense reagents to inhibit miRNAs by conjugating 2′-O-methyl oligoribonucleotide with dextran and rhodamine. The dextran-conjugated antisense reagents can be conveniently introduced into the germline of adult hermaphrodites and are transmitted to their progeny, where they efficiently inhibit a targeted miRNA in different tissues, including the hypodermis, the vulva and the nervous system (Table 1). A number of control experiments confirmed that these antisense reagents selectively target their corresponding miRNAs with high sequence specificity. Further, these reagents can be used combinatorially to inhibit more than one miRNAs in the same animal, thus providing a convenient approach to examine genetic interactions that involve miRNA, especially when combined with numerous mutants or reporter stains available.
Table 1. Inhibit miRNAs in C. elegans using dextran-conjugated antisense reagents.
|RhD-as-2’OMelin-4||50||lin-4||N2||Egl, vulvaless, no alae during L4 lethargus||100|
|VT1367||No col-19::GFP expression 10 hr post-L4||100|
|JR667||Seam cells repeat L2 fate in L3||100|
|RhD-as-2’OMelet–7||50||let-7||N2||Bursting vulva, no alae||51±6|
|RhD-as-2’OMelsy-6||3||lsy-6||OH3191||No gcy-7::gfp expression in ASEL||98±2|
|OH3192||gcy-5::gfp expression in ASEL||94±6|
|RhD-as-2’OMemiR-42||3||miR-42||MT14119||Embryonic or L1 lethal||77±13|
|OH3192||Egl, gcy-5::gfp expression in ASEL||100|
a “RhD” denotes rhodamine labeled dextran; “as-2’OMe” represents antisense 2’-O-methyl oligoribonucleotide.
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