A Feasible Method For Caenorhabditis Elegans Culture And Its Application In Anticancer Drug Screening

Caenorhabditis elegans strain of MT2124 contains a mutant let-60/ras,which showed multivulva phenotype generally with 93%penetrance when is cultured on NGM plate.However,it occures semi-dominant under some uncertain conditions with about 17%more or less of hermaphroditic worms are Muv.Hence when nematodes of MT2124 are used in screening anticancer drug candidates which can inhibit the excessive activation of the ras oncogene,their uncertain phenotypes lead to questionable results.C.elegans ras mutant of MT2124 was used to explore a feasible method for its culture in our experiments.The object was to optimize MT2124 culture protocol to obtain animal populations with stable Muv phenotype for their further application on anticancer drug candidates screening.Liquid culture with food resource of live or killed E.coli OP50,and CeHR axenic culture methods were tested in our experiment.A feasible method was finally established to cultivate the C.elegans ras mutant MT2124:0.4g of live E.coli OP50 and 10000 nematode individuals at Ll stage were added to 15 mL S medium in a flask,then the flask was shaken at 20?,130rpm till nematode larvae got mature.After synchronized,the number of L1 larvae is higher than any other methods,and the Muv phenotype of each batch is stable.C.elegans of MT2124 L1 larvae obtained by this kind of method was used to evaluate the activity of ShengMai san(SM)for inhibiting the ras gene excessive activation.The experiment results showed that SM could significantly suppress the Muv phenotype of MT2124,but it could not revert the Muv phenotype of MT8189 and CB1275,indicating that SM suppress ras excessive activation is mechanism based rather than a non-specific cytotoxicity,and C.elegans with wild ras copy was not sensitive to SM.The activity of number 1 extracts and number 2 extracts for inhibiting the excessive activation of ras gene was also tested in our experiment,both of them had a high activity,and when the activity of the extracts tested were equivalent,the number 1 extracts had higher toxicity,in contrast,number 2 extracts showed no toxicity.Additionally,after further purified,the activity of number 2 extracts was higher than that number 1 extracts.