Shotgun Antisense

Shotgun antisense allows us to identify an interesting mutant in the morning and start sequencing the gene’s DNA in the afternoon. We developed this technique for mutagenesis of Dictyostelium cells, based on the observation that expression of antisense RNAs works well in this system to repress expression of selected gene products.

The figure shows a vector for expressing antisense RNAs; these will hybridize to mRNAs expressed by the transformed cell, preventing their expression. The cDNA library can be made from any selected developmental stage or cell-type, and the promoter can also be developmental-stage or cell-type specific. Transformed cells are first selected with a drug that kills untransformed cells (in this case, neomycin, which will kill untransformed cells, but transformed cells are protected because they have the neoR gene). Transformed cells typically take up only one transformation plasmid, so each transformed cell has a different antisense RNA, preventing expression of a single gene (or multigene family).  After screening this pool of mutated cells and selecting a mutant of interest, a PCR reaction using the indicated primers will yield the cDNA fragment corresponding to the gene whose expression is being suppressed in the mutant of interest.

For instance, for one project we were interested in finding genes expressed in vegetative growth that affect cell-type choice during late development, so we used a vegetative promoter and a vegetative cDNA library.

References

  • Crowley, T.E., Nellen, W., Gomer, R.H., and Firtel, R.A.  Phenocopy of discoidin I- minus mutants by anti-sense transformation in Dictyostelium.  Cell 43, 633-641 (1985).
  • Spann, T.P., Brock, D.A., Lindsey, D.F., Wood, S.A., and Gomer, R.H. Mutagenesis and gene identification in Dictyostelium by shotgun antisense.  Natl. Acad. Sci. USA  93, 5003-5007 (1996).
  • Wood, S.A., Ammann, R.R., Brock, D.A., Li, L., Spann, T.P., Gomer, R.H.  RtoA links initial cell type choice to the cell cycle in Dictyostelium.  Development 122, 3677-3685 (1996).
  • Brock, D.A., Buczynski, F., Spann, T.P., Wood, S.A., Cardelli, J., and Gomer, R.H. A Dictyostelium mutant with defective aggregate size determination.  Development 122, 2569-2578 (1996).