Fast cloning inverted repeats for RNA interference

Abstract

Double-stranded RNA (dsRNA) can induce post-transcriptional gene silencing in a wide variety of organisms. Commonly, inverted repeats are used to produce dsRNA to silence genes of interest. However, cloning inverted repeats still remains a rate-limiting step for widely applying this technique. Here we describe a pGEM-T-based vector, pGEM-WIZ, designed to produce inverted repeats for any Drosophila gene. pGEM-WIZ has a high efficiency in assembling inverted repeats and the repeats in this vector are stable in regular Escherichia coli strains. Furthermore, we have developed a method for rapid selection of clones with an inverted repeat based on size and relative copy number of the vector with or without an insert. This method further eases the cloning process. The inverted repeat cassette assembled in pGEM-WIZ can be easily transferred to commonly available expression vectors suitable for stably expressing inverted repeats in vitro and in vivo.

FIGURE 1.

Schematic representation of the pGEM-S1 and pGEM-WIZ vectors. (A) pGEM-S1 was constructed by circularizing pGEM-T (Promega). As a result, an additional six unique sites are present in pGEM-S1, marked by asterisks (*). (B) pGEM-WIZ was constructed by shuttling the intron 2 of the Drosophila gene white from pWIZ into pGEM-S1. The white intron contains consensus splicing sites: 5′-splice site (AG↑GTRAGT) and 3′-splice site (TTTYYYYTNCAG↑RT), where ↑ stands for the splice site; R for A or G; Y for C or T; and N for any base. MCS=multiple cloning sites.

FIGURE 2.

Experimental strategy for cloning inverted repeats using pGEM-WIZ. For clarity, assembling an inverted repeat for the Drosophila gene hibris is given as an example. Please note, to form a functional inverted repeat, the two fragments can be arranged in either a head–head or a tail–tail fashion. w=intron 2 of the white gene.

FIGURE 3.

Rapid selection for clones with an inverted repeat based on size and relative amount of plasmid DNA. Selection of an inverted repeat for the Drosophila gene hibris is shown here as an example. A total of 40 μL of plasmid DNA was isolated from 0.8 mL of a 4 mL overnight culture by a modified STET-lysozyme method (see Materials and Methods). Four microliters of DNA samples were loaded on a 1% agarose gel containing 0.5 μg/mL ethidium bromide. Fill arrows point to the clones with an insert that run at a position corresponding to a higher molecular weight, while open arrows point to the control vectors. (A) A 504-bp fragment verified by sequencing was inserted into pGEM-WIZ at an AvrII site. The clones (1,2,3,5,6) with an insert were unambiguously identified by size compared with the control pGEM-WIZ alone (r). (B) The same fragment was inserted into the NheI site of pGEM-WIZ that contains the first insert. The clones with double inserts were selected by size (1,3,4,5,6). Among them, the ones with significantly weaker intensity of the bands (3,4,5) were identified to contain an inverted repeat. pGEM-WIZ with one insert was used as a control (r). (C) The whole inverted repeat was excised from pGEM-WIZ with XhoI/XbaI and ligated into pUAST. Clones with an inverted repeat were identified by size (1,2,4,5,6). pUAST was used as a control (r). All selected clones have been confirmed by restriction mapping.