A heat-shock expression system in Sf-9 insect cells (Sf-9 cell)

Description

Infection by many viruses triggers apoptosis. This can benefit either the virus or the host, and accordingly many hosts and virus have evolved mechanisms and counter mechanisms designed to promote and inhibit apoptosis. Thanks to the methods described here, several apoptosis-related genes have been identified in both shrimp and WSSV, and we expect that more related genes will be identified in the future. This section introduces three methods for testing the anti- or pro-apoptosis activities of genes in Sf-9 cells.

Brief protocol

Viability assays for pro-apoptosis proteins

Sf-9 cells seeded in six-well plates (5 × 10⁵ cells/well) are transfected with the appropriate plasmids using Cellfectin reagent (Invitrogen). After transfection for 16-18 h, the cells are heat shocked in a 42℃ water bath for 30 min and then returned to 27℃. At 5 h after heat shock, the media are removed, and the cells are resuspended in 1 ml PBS containing 0.04% trypan blue with a rubber policeman. The viable intact cells are counted with a hemocytometer, and eight grids are counted for each sample. The mean and standard deviation are calculated from three replicate samples. Relative percent viability is calculated by comparing the number of viable cells transfected with various expression plasmids with the number of viable cells transfected with the control plasmid. The relative percent viability for cells transfected with the control plasmid is set at 100%.

Actinomycin D assay for anti-apoptosis proteins

Sf-9 cells seeded in six-well plates (1 x 10⁶ cells/well) are transfected and heat shocked to induce protein expression according to above description. At 4 h after heat shock, the media are removed and replaced with fresh media containing 150 ng/ml of actinomcyin D (ActD). At 14-16 h after addition of ActD, the media are removed and the cells are washed with phosphate buffered saline (PBS; 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) and then stained with 0.04% trypan blue in PBS. The viable, intact cells are counted using a bright-field microscope equipped with a digital camera and AutoCell software. The total number of viable cells is determined from five evenly distributed fields of view at 200x magnification. The viability of the cells used to express the indicated proteins is calculated by dividing the total number of viable cells remaining after drug treatment by the total number of viable cells in control transfections at the time of drug addition.

DNA ladder analysis

Apoptotic DNA fragments are isolated according to the method published by Herrmann et al. Sf-9 cells in six-well plates (1 × 10⁶ cells/well) are transfected and heat shocked to induce protein expression as described above. Overnight after heat shock, the cells and apoptotic bodies are pooled and suspended in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 20 mM EDTA, and 1% NP-40. After centrifugation for 5 min at 1600 X g, the supernatant is collected and the pellet is extracted again with lysis buffer. Both supernatants are pooled, brought to 1 % SDS and to 5 μg/μl RNase A and incubated at 56°C for 2 h. Proteinase K is added to a final concentration of 2.5μg/μl and incubation continued at 37°C for at least 2 h. The ladder DNA is precipitated with 1/2 vol. of 10 M ammonium acetate and 2.5 vol. of ethanol, then re-dissolved in gel loading buffer and analyzed by agarose gel electrophoresis.

References

1. Herrmann M, Lorenz HM, Voll R, Grunke M, Woith W, Kalden JR. (1994) A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Res 22: 5506-5507.
2. Khanobdee, K., Soowannayan, C., Flegel, T.W., Ubol, S., Withyachumnarnkul, B. (2002) Evidence for apoptosis correlated with mortality in the giant black tiger shrimp Penaeus monodon infected with yellow head virus. Dis Aquat Org 48: 79-90.
3. Chayaburakul, K., Lightner, D.V., Sriurairattana, S., Nelson, K.T., Withyachumnarnkul, B. (2005) Different responses to infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Penaeus monodon and P. vannamei. Dis Aquat Org 67: 191-200.
4. Sahtout, A.H., Hassan, M.D., Shariff, M. (2001) DNA fragmentation, an indicator of apoptosis, in cultured black tiger shrimp Penaeus monodon infected with white spot syndrome virus (WSSV). Dis Aquat Org 44: 155-159.
5. Wongprasert, K., Khanobdee, K., Glunukarn, S.S., Meeratana, P., Withyachumnarnkul, B. (2003) Time-course and levels of apoptosis in various tissues of black tiger shrimp Penaeus monodon infected with white-spot syndrome virus. Dis Aquat Org 55: 3-10.

Some of our publications that have used this platform

1. Leu JH, Kuo YC, Kou GH, Lo CF. (2008) Molecular cloning and characterization of an inhibitor of apoptosis protein (IAP) from the tiger shrimp, Penaeus monodon. Dev Comp Immunol 32: 121-133.
2. Leu JH, Wang HC, Kou GH, Lo CF. (2008) Penaeus monodon caspase is targeted by a white spot syndrome virus anti-apoptosis protein. Dev Comp Immunol 32: 476-486.