Targeting gene expression to the female larval fat body of transgenic Aedes aegypti mosquitoes.

As the fat body is a critical tissue for mosquito development, metamorphosis, immune and reproductive system function, the characterization of regulatory modules targeting gene expression to the female mosquito fat body at distinct life stages is much needed for multiple, varied strategies for controlling vector-borne diseases such as dengue and malaria. The hexameric storage protein, Hexamerin-1.2, of the mosquito Aedes atropalpus is female-specific and uniquely expressed in the fat body of fourth instar larvae and young adults. We have identified in the Hex-1.2 gene, a short regulatory module that directs female-, tissue-, and stage-specific lacZ reporter gene expression using a heterologous promoter in transgenic lines of the dengue vector Aedes aegypti. Male transgenic larvae and pupae of one line expressed no Escherichia coli ß-galactosidase or transgene product; in two other lines reporter gene activity was highly female-biased. All transgenic lines expressed the reporter only in the fat body; however, lacZ mRNA levels were no different in males and females at any stage examined, suggesting that the gene regulatory module drives female-specific expression by post-transcriptional regulation in the heterologous mosquito. This regulatory element from the Hex-1.2 gene thus provides a new molecular tool for transgenic mosquito control as well as functional genetic analysis in aedine mosquitoes.

piggyBac internal sequences are necessary for efficient transformation of target genomes.

A previously reported piggyBac minimal sequence cartridge, which is capable of efficient transposition in embryo interplasmid transposition assays, failed to produce transformants at a significant frequency in Drosophila melanogaster compared with full-length or less extensive internal deletion constructs. We have re-examined the importance of these internal domain (ID) sequences for germline transformation using a PCR strategy that effectively adds increasing lengths of ID sequences to each terminus. A series of these piggyBac ID synthetic deletion plasmids containing the 3xP3-ECFP marker gene are compared for germline transformation of D. melanogaster. Our analyses identify a minimal sequence configuration that is sufficient for movement of piggyBac vectored sequences from plasmids into the insect genome. Southern hybridizations confirm the presence of the piggyBac transposon sequences, and insertion site analyses confirm these integrations target TTAA sites. The results verify that ID sequences adjacent to the 5' and 3' terminal repeat domains are crucial for effective germline transformation with piggyBac even though they are not required for excision or interplasmid transposition. Using this information we reconstructed an inverted repeat cartridge, ITR1.1k, and a minimal piggyBac transposon vector, pXL-BacII-ECFP, each of which contains these identified ID sequences in addition to the terminal repeat configuration previously described as essential for mobility. We confirm in independent experiments that these new minimal constructs yield transformation frequencies similar to the control piggyBac vector. Sequencing analyses of our constructs verify the position and the source of a point mutation within the 3' internal repeat sequence of our vectors that has no apparent effect on transformation efficiency.

Polyubiquitin-regulated DsRed marker for transgenic insects.

Genetic transformation of most insect systems requires dominant-acting markers that do not depend on reverting a mutant phenotype in a host strain, andfor this purpose GFP has proven to be useful in several insect orders. However, detection of multiple transgenes and reporters for gene expression requires the development of new visible markers that can be unambiguously detected when co-expressed with GFP The DsRed fluorescentprotein has spectral characteristics that are most distinct from GFP and GFP variants, and we have explored the use of DsRed as a selectable marker for piggyBac transformation in Drosophila melanogaster and its use as a reporter when co-expressed with GFP. Transformants marked with polyubiquitin-regulated DsRed1 were detected throughout development at a relatively high frequency, and they exhibited brighter fluorescence than transformants marked with EGFP. The use of a Texas Red filter set eliminated detection of EGFP fluorescence and autofluorescence, and DsRed expressedfrom a reporter construct could be unambiguously detected when co-expressed with EGFP DsRed should prove to be a highly efficient marker system for the selection of transformant insects and as a reporter in gene expression studies.

A randomized, double-blind, crossover clinical trial of diclofenac plus misoprostol versus acetaminophen in patients with osteoarthritis of the hip or knee.

OBJECTIVE: To perform a randomized, double-blind, crossover clinical trial of diclofenac + misoprostol versus acetaminophen in ambulatory patients with osteoarthritis of the hip or knee. METHODS: Patients in 12 ambulatory care settings were eligible if they were age >40 years and if they had Kellgren/Lawrence radiographic grade 2-4 osteoarthritis of the knee or hip and a score of > or =30 mm on a 100-mm visual analog pain scale. Patients were randomized to one of two groups, 75 mg diclofenac + 200 microg misoprostol twice daily or 1,000 mg acetaminophen 4 times daily (each for 6 weeks), and were then crossed over to the other treatment for 6 weeks. A placebo was included in each treatment regimen to enable double blinding. The primary outcome measures were the Western Ontario and McMaster Universities Osteoarthritis Index and the visual analog pain scale of the Multidimensional Health Assessment Questionnaire. Safety was assessed using a standard form to review adverse events. RESULTS: We enrolled 227 patients, of whom 218 provided data for the first treatment period and 181 provided data for both treatment periods. Significantly higher levels of improvement in the primary outcomes were seen for diclofenac + misoprostol than for acetaminophen (P < 0.001). Adverse events were more common when patients took diclofenac + misoprostol (P = 0.046). Diclofenac + misoprostol was rated as "better" or "much better" by 57% of the 174 patients who provided such ratings for both treatment periods, while acetaminophen was rated as "better" or "much better" by 20% of these patients, and 22% reported no difference (P < 0.001). Differences favoring diclofenac + misoprostol over acetaminophen were greater in patients with more severe osteoarthritis according to baseline pain scores, radiographs, or number of involved joints. CONCLUSION: Patients with osteoarthritis of the hip or knee had significantly greater improvements in pain scores over 6 weeks with diclofenac + misoprostol than with acetaminophen, although patients with mild osteoarthritis had similar improvements with both drugs. Acetaminophen was associated with fewer adverse events.

Transformation of the Caribbean fruit fly, Anastrepha suspensa, with a piggyBac vector marked with polyubiquitin-regulated GFP.

Germ-line transformation was achieved in the Caribbean fruit fly, Anastrepha suspensa, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Four transgenic G(0) lines were selected exhibiting unambiguous GFP expression. Southern hybridization indicated the presence of one to four integrations in each of the transgenic lines with two integrations verified as piggyBac-mediated by sequencing their insertion sites. Fluorescence was detectable throughout development, and in adults was most intense from the thoracic flight muscle. Although adult cuticle quenched fluorescence, GFP was routinely detectable in the thorax. A quantitative spectrofluorometric assay was developed for GFP fluorescence that indicated differing levels of fluorescence among the transgenic lines, suggesting some level of position effect variegation/suppression. These results are encouraging for the use of this marker system in insect species not amenable to mutation-based visible markers. Together with the piggyBac vector, a transformation system is presented that has the potential to be universally applicable in insect species.

Germline transformation of Drosophila melanogaster with the piggyBac transposon vector.

Germline transformation of Drosophila melanogaster was attempted with the piggyBac gene-transfer system from the cabbage looper moth, Trichoplusia ni. Using a self-regulated transposase helper and a white marked vector, a transformation frequency of 1-3% per fertile G0 was obtained, similar to that previously achieved in the medfly. Use of an hsp70-regulated helper increased this frequency more than eight-fold. Transformation with a vector marked with white and green fluorescent protein (GFP) under polyubiquitin-nuclear localizing sequence regulation yielded seventy G1 transformants which all expressed GFP, but only twenty-seven of these expressed eye pigmentation that would have allowed their selection based on white+ expression. PiggyBac transformation in two distantly related dipteran species and efficient expression of the gfp marker supports the potential use of this system in other dipterans, and perhaps insects in general.