SMG-ly knocking out gene expression in specific cells: an educational primer for use with "a novel strategy for cell-autonomous gene knockdown in caenorhabditis elegans defines a cell-specific function for the G-protein subunit GOA-1"

Abstract

A recent article by Maher et al. in GENETICS introduces an alternative approach to cell-type-specific gene knockdown in Caenorhabditis elegans, using nonsense-mediated decay. This strategy has the potential to be applicable to other organisms (this strategy requires that animals can survive without nonsense-mediated decay-not all can). This Primer article provides a guide and resource for educators and students by describing different gene knockdown methodologies, by assisting with the technically difficult portions of the Maher et al. article, and by providing conceptual questions relating to the article.

Keywords: cell-specific gene expression; education; nonsense-mediated decay (NMD).

Figure 1

Methods for knocking out a gene or its function in specific cells. (A) Mosaic analysis by loss of a chromosomal fragment, used primarily in C. elegans. A cell with the duplication has a wild-type phenotype but loses the duplication at random during mitosis. This results in patches or clones of cells with a mutant genotype. The embryo on the left has lost the duplication in the rectangular cell, so its descendants have a mutant genotype. The embryo on the right has lost the duplication in the oval cell, so its descendants have a mutant genotype. The investigator searches within the population for individuals with the mutant genotype in the cells of interest. (B) Cre-lox excisions or rearrangements, used primarily in mice. In this example, the Cre recombinase is under the control of the insulin regulatory region, so it will be transcribed only in the pancreas. The gene of interest has loxP sites flanking exons 4 and 5. When Cre is expressed in the pancreas, recombination between the two loxP sites results in the deletion of exons 4 and 5, inactivating the gene. In other cells, Cre is not expressed and no deletion occurs. (C) RNAi using a cell-specific promoter. A region of the gene of interest is placed under the control of a cell-specific promoter, often from a microRNA gene expressed in that cell type. An inverted region of the gene of interest is cloned downstream of it. When the construct is transcribed in those cells, a hairpin of the transcript forms with the siRNA sequences as its stem. This is processed to the dsRNA to make the functional siRNA molecule in those cells.

Figure 2

A summary of nonsense-mediated decay (NMD). A transcript has its normal UGA stop codon but a nonsense mutation results in a premature termination codon (PTC). A complex of UBF1, UBF2, and UBF3 forms at or near the PTC. Degradation of the transcript is regulated by a cycle of phosphorylation (regulated by SMG-1) and dephosphorylation (regulated by SMG-5, SMG-7, and SMG-6). All of these seven genes are necessary for nonsense-mediated decay.

Figure 3

The use of NMD for cell-specific gene knockouts. The smg-5+ gene, which is needed for NMD, is integrated under the control of the tph-1 regulatory region, which results in transcription in only four neurons, including the HSNs. The goa-1+ gene with the termination tag from let-858 is integrated in the genome as well. In the HSNs, smg-5 is transcribed, so NMD occurs, and the goa-1 transcript with the termination tag is degraded. This results in the failure to rescue the goa-1 mutant phenotype in the HSNs. In other neurons, smg-5 is not transcribed, so there is no NMD, and the goa-1 transcript persists. In these cells, the goa-1 mutant phenotype is rescued.