Alternative mRNA splice variants in Drosophila 



JESO Volume 139, 2008 



Methods and Materials 



Cell Line and Viral Infection 



DL2 cells were obtained from ATCC, and cultured in Schneider's Drosophila 

 media (SDM) supplemented with 9% heat inactivated fetal bovine serum and 100 ug/mL 

 kanamycin (henceforth referred to as media) (Schneider 1972). DL2 cells were grown 

 at 22°C without CO, and subcultured every 3 days. The cells were infected with Flock 

 House Virus (FHV) (gift from Dr. David Miller) as follows: Subconfluent DL2 were gently 

 dislodged and pelleted by centrifugation (300g). Cells were then resuspended in media and 

 counted with a haemocytometer. Cell viability was determined with Trypan blue exclusion 

 to ensure viability greater than 90%. Cell concentration was adjusted to 10 7 cells/ml with 

 SDM and FHV added at a multiplicity of infection (MOI) of 1 0. Following inoculation cells 

 were incubated at room temperature with occasional gentle mixing for 1 h. Cell density was 

 then adjusted to 10 6 cells per ml with SDM, and incubated at 22°C without C0 2 exchange. A 

 mock infection was performed on a replicate culture. The mock infected cells were treated 

 as described above except the inoculum did not contain FHV. The cell morphology in all 

 cultures was periodically monitored by light microscopy up to 72 h post infection. Under 

 our infection protocol conditions, 95% of DL2 cells are infected by 8 h post inoculation and 

 maximum viral RNA replication takes place between 4 and 16 h post inoculation (Dr. David 

 Miller, pers. comm.). 



RNA Extraction and RT-PCR 



Total RNA was extracted according to the manufacturer's instructions using 

 QIAGEN Easy RNA extraction kit from 2xl0 6 cells at 8, 12, 20 and 44 h post infection 

 from the infected cells and at 8 h from the mock-infected control. The RNA was then 

 treated with DNAse for 30 min at 37 °C followed by 1 h incubation at 80 °C to degrade 

 genomic DNA. Oggl was amplified from RNA using QIAGEN one step RT-PCR according 

 to the manufacturer's instructions. The primers used were forward 5'- CGGGATCCAT 

 GAAGGCTGTTTTAC and a reverse 5 ' -TAG ATAAG AT CACTTTTTAGG. 



Splice Variant Characterization and Frequency Calculation 



PCR products were cloned into pGEM-T easy vector (Promega) and individual 

 Oggl transcripts were characterized by PCR, Sail restriction endonuclease analysis, and 

 DNA sequencing as previously described (Skandalis et al. 2004). Splice variant frequency 

 was calculated as the fraction of the transcripts characterized that exhibited an alternative 

 splicing pattern and it was reported as a percentage. 



Results 



Cell Morphology and Cytopathic Effect 



FHV inoculated and mock infected DL2 cells were periodically monitored by 

 light microscopy for morphological changes. DL2 cells adhered loosely to tissue culture 

 plates during growth as previously observed (Schneider 1972). Adherence to culture 

 plates decreased with time following FHV inoculation and gross morphological changes 



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