Short-hairpin RNA expressed from polymerase III promoters mediates RNA interference in mosquito cells.

Putative U6snRNA polymerase III (PolIII) promoters were cloned from the Anopheles gambiae and Aedes aegypti genomes. The PolIII promoters were tested for their ability to express short-hairpin RNA (shRNA) targeted to firefly luciferase and to mediate RNA interference (RNAi) knockdown of a co-transfected luciferase reporter gene vector in AG-55 Anopheles gambiae and ATC-10 Aedes aegypti cells. Promoters capable of silencing expression of the co-transfected luciferase plasmid by up to 95% in AG-55 cells and up to 75% in ATC-10 cells were identified. RNase protection experiments allowed detection of the 19 nt luciferase short-interfering RNA (siRNA) in transfected cells. These findings indicate that mosquito U6snRNA gene promoters can be used for production of shRNA to induce the RNAi response in mosquito cells.

Aedes aegypti transducing densovirus pathogenesis and expression in Aedes aegypti and Anopheles gambiae larvae.

Aedes aegypti densovirus (AeDNV) is a small DNA virus that has been developed into an expression and transducing vector for mosquitoes [Afanasiev et al. (1994) Exp Parasitol 79: 322-339; Afanasiev et al. (1999) Virology 257: 62-72; Carlson et al. (2000) Insect Transgenesis: Methods and Applications (Handler, A.M. & James, A.A., eds), pp. 139-159. CRC Press, Boca Raton]. Virions carrying a recombinant genome expressing the GFP gene were used to characterize the pathogenesis of the virus in 255 individual Aedes aegypti larvae. The anal papillae of the larvae were the primary site of infection confirming previous observations (Afanasiev etal., 1999; Allen-Muira et al. (1999) Virology 257: 54-61). GFP expression was observed in most cases to spread from the anal papillae to cells of the fat body, and subsequently to many other tissues including muscle fibers and nerves. Infected anal papillae were also observed to shrink, or melanize and subsequently fall off in a virus dependent manner. Three to four day-old larvae were less susceptible to viral infection and, if infected, were more likely to survive into adulthood, with 14% of them still expressing GFP as adults. Higher salt concentrations of 0.10-0.15 M inhibited viral infection. Anopheles gambiae larvae also showed infection of the anal papillae (17%) but subsequent viral dissemination did not occur. The persistence of the reporter gene expression into adulthood of Aedes aegypti indicates that transduction of mosquito larvae with recombinant AeDNV may be a means of introducing a gene of interest into a mosquito population for transient expression.

Leukaemogenesis by the delta Mo + SV Moloney murine leukaemia virus (M-MuLV) variant in E mu pim-1 transgenic mice: high frequency of recombination with a solo endogenous M-MuLV LTR in vivo.

We previously described an enhancer variant of Moloney murine leukaemia virus (M-MuLV), delta Mo + SV M-MuLV, in which the enhancers of MuLV have been deleted and replaced with the enhancers of the simian virus 40 (SV40). When this virus is injected into neonatal NIH Swiss mice, pre-B and B-lymphoblastic lymphomas develop with a latency of 17 months. Van Lohuizen et al. (1989) described a line of transgenic mice that carry an activated pim-1 proto-oncogene transgene (E mu pim-1). They also reported that E mu pim-1 transgenic mice show greatly accelerated lymphoma development when infected with wild-type M-MuLV at birth. In these experiments, neonatal E mu pim-1 transgenic mice were infected intraperitoneally with delta Mo + SV M-MuLV. Marked acceleration of T-lymphoid leukaemia was seen. However, 10 of the 11 tumours analysed were found to be negative for the SV40 enhancers, but they still contained M-MuLV DNA as measured by Southern blot analysis. The LTRs on viruses cloned from two such tumours (as well as on virus recovered by infection onto NIH 3T3 cells) were characterized by PCR amplification, molecular cloning and sequence analysis. The LTR's from the two tumours were identical to each other and were distinct from both the delta Mo + SV M-MuLV and wild-type M-MuLV LTRs. However, they were identical to a rearranged solo M-MuLV LTR present in the E mu pim-1 transgene. These results indicate that the recombination in vivo between delta Mo + SV M-MuLV and the E mu pim-1 transgene yielded a replication-competent and pathogenic virus at high efficiency. This is the first report of in vivo recombination between an exogenous MuLV and a solo endogenous LTR.