Search

The insect steroid hormone 20-hydroxyecdysone initiates a cascade of regulatory events in a temporal and tissue-specific manner by first binding to a complex of an ecdysone receptor (EcR) protein and a ultraspiracle protein. Using an antisense (As) ribonucleic acid approach, we show that disruption of EcR expression in transfected C7-10 cells from the mosquito Aedes albopictus affects survival and growth. From stably transfected cells, we recovered a new isoform of A. albopictus AalEcRa, which is named AalEcRb. The deduced amino acid sequence of AalEcRb was almost identical to that of AalEcRa, with the exception of a seven amino acid sequence near the C-terminus. Using polymerase chain reaction followed by restriction enzyme analysis, we found that AalEcRa is the predominant species expressed by wild-type C7-10 cells, while cells transfected with As-EcR expressed both isoforms at approximately equal levels.

Novel approaches to control of vector-borne disease include potential use of transgenic insects, in which molecular mechanisms will be induced to prevent transmission of pathogenic organisms. The infrastructure essential to this technology includes the cloning of essential genes from vector insects, and the development of efficient transformation strategies. In this study, we use a continuous mosquito (Aedes albopictus) cell line and a cloned mosquito dihydrofolate reductase gene to demonstrate a transgenic approach that may be used to select for the presence or absence of particular gene functions in transfected cells. Plasmids containing the dihydrofolate reductase gene in sense and antisense orientation, under the regulation of a temperature-inducible promoter, were expressed in stably transfected mosquito cells. At the normal growth temperature of 28 degrees C, or after mild heat induction at 34 degrees C, expression of the dihydrofolate reductase construct in sense orientation had little effect on cell growth. In contrast, recovery of clones transfected with the antisense construct was reduced, and induction of antisense transcripts at 34 degrees C further compromised cell growth and viability. Clones transfected with the sense construct retained significantly higher copy numbers of foreign DNA than did cells transfected with the antisense construct. These studies provide a basis for use of sense and antisense dihydrofolate reductase constructs to recover transfected mosquito cells with specific desired phenotypes, based on the relative expression of cloned genes of interest.

An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10-15 days in the presence of selective medium containing 1 microM methotrexate. The transformed clones contained an estimated 100-500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, but in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture.

Transient expression of a heat-shock protein-chloramphenicol acetyltransferase (hsp-CAT) recombinant plasmid was used to define the parameters that influence transfection of cultured mosquito cells using DNA-calcium phosphate coprecipitates. The efficiency of the calcium phosphate procedure was strongly influenced by the growth state of recipient cells, and by the temperature at which the coprecipitate was prepared. Under optimal conditions, which included formation of the DNA-calcium phosphate coprecipitate at 50 degrees C, transfection frequencies were up to tenfold higher than those obtained using the previously described polybrene procedure.

We have introduced hsp-cat plasmid DNA into Spodoptera frugiperda (Lepidoptera: Noctuidae) cells by transfection with purified DNA (1 to 48 micrograms/ml) mixed with the polycation polybrene (100 micrograms/ml) in serum-free Grace's medium. The hsp-cat construct contains a gene coding for the bacterial enzyme chloramphenicol acetyltransferase (CAT), whose expression is controlled by a promoter derived from a Drosophila heat shock (hsp) gene. Expression of CAT activity in transfected Spodoptera cells was induced by a 2-h heat shock at 43 degrees C. The temperature of the heat shock was based on conditions that maximized the expression of endogenous heat shock protein genes in these cells. CAT activity was maximal in cells that were exposed to the heat shock 2 d after transfection; by 4 d, activity was diminished, and little activity was detectable after 6 d. Transfection frequencies, which varied with DNA concentration and ranged as high as 6000 per million cells, were determined using a histochemical staining procedure.

The polycation 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) is superior to calcium phosphate for the introduction of purified DNA into cultured Aedes albopictus (mosquito) cells. Adsorption of the polybrene-DNA complex to mosquito cells was essentially linear for 6 h. However, the rate of adsorption of DNA increased when the DNA-polybrene mixture was preincubated for several hours prior to addition to cells. A recombinant plasmid carrying an inducible chloramphenicol acetyltransferase gene under the control of a Drosophila heat shock protein (hsp) promoter was used to show that expression of transfected DNA was highest when cells were treated with a freshly prepared polybrene-DNA mixture. Optimal expression was observed in cells transfected with 4-13 micrograms of DNA per 10(6) cells; transfection with 24 micrograms of DNA resulted in reduced CAT expression. Variation in the polybrene-DNA ratio improved transfection with high levels of DNA. In mosquito cells, CAT expression was independent of DNA methylation.