RNAi in the Red Flour Beetle (Tribolium)

Abstract

INTRODUCTIONTribolium castaneum is exceptionally amenable to gene knockdown by RNA interference (RNAi) which, in this insect, is systemic (spreading throughout the organism and to the next generation), highly penetrant, and able to phenocopy genetic null phenotypes. Hence, any gene function can be knocked down at any stage in (apparently) all tissues upon injection of double-stranded RNA (dsRNA). The RNAi effect is elicited both in the injected animal and, if female pupae or adults have been injected, transferred to the offspring. Embryonic RNAi (eRNAi) usually generates the strongest phenotypes in the injected individual, but suffers from elevated lethality caused by injection injury. Pupal RNAi (pRNAi), in which female pupae are injected and phenotypes scored in the offspring, is the easiest to perform. However, in some cases, the knockdown of a gene leads to sterility of the injected female. This problem can be circumvented in many cases by injecting adult females (aRNAi) or using eRNAi. In order to interfere with processes during metamorphosis, injection into last-stage larvae is used (lRNAi). Up to two genes in a single experiment have been successfully knocked down via RNAi. The inclusion of more than two genes usually leads to a dilution effect, which lowers phenotypic strength. This protocol describes the production of dsRNA from a polymerase chain reaction (PCR) template, injection procedures for each Tribolium life stage, and important controls for effective analysis.

Hox genes require homothorax and extradenticle for body wall identity specification but not for appendage identity specification during metamorphosis of Tribolium castaneum

“…The flour beetle Tribolium castaneum has emerged as an attractive system for studying both embryogenesis and metamorphosis in insects (Posnien et al, 2009;Tomoyasu and Denell, 2004). This species is especially suitable for investigating the relationship between embryonic and metamorphic segmental identity specification because, unlike Drosophila larvae, Tribolium larvae possess ventral appendages, which are repatterned during metamorphosis to form the adult appendages.…”

Efficient CRISPR-mediated gene targeting and transgene replacement in the beetleTribolium castaneum

“…A 2-kb fragment containing the largest exon was cloned from genomic DNA, using primers AG001 and AG002 (supplementary material Table S1), into pGEM-T-Easy (Promega). Double-stranded RNA was produced as described previously (Posnien et al, 2009). …”

“…Starting with classic genetic screens conducted in the 1980s (Beeman et al, 1989;Sulston and Anderson, 1996), the Tribolium research community has grown, bringing a plethora of genetic tools and approaches to this species. These approaches include transgenesis (Berghammer et al, 1999(Berghammer et al, , 2009Lorenzen et al, 2003;Pavlopoulos et al, 2004), RNAimediated knockdown (Bucher et al, 2002;Posnien et al, 2009), enhancer trapping (Lorenzen et al, 2003;Trauner et al, 2009), heatshock and GAL4/UAS-mediated mis-expression (Schinko et al, 2012(Schinko et al, , 2010 and live imaging (Benton et al, 2013;Sarrazin et al, 2012;Strobl and Stelzer, 2014). Moreover, the Tribolium genome is sequenced and well annotated (Kim et al, 2010;Richards et al, 2008).…”

Triboliumembryo morphogenesis

“…7,8 Starting with the seminal work by the geneticist Alexander Sokoloff in the 1960s, 9 Tribolium has become an increasingly popular and powerful system for developmental genetic studies. Tribolium lends itself to forward and reverse genetic approaches, [10][11][12][13] is amenable to transgenic manipulations, [14][15][16][17] and is supported by a sequenced genome and other resources for genome-wide research.…”