RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

Summary

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

Abstract

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways¹. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles.

Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi². This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

Protocol

1. Screening strain construction

The careful design of the screening strain is critical for the success of the screen and has been described elsewhere³. For some researchers, using a strain that expresses a visible product from a transgene is needed for the experiment. Many strains harboring integrated transgenes are available from the CGC or individual researchers. If a transgenic strain is required for the screen but is not available, then it can be generated using a published method like bombardment⁴, UV/TMP⁵, or Mos transposon insertion⁶. In order to visualize our protein of interest, we inserte....

Discussion

The RNAi screening method presented here enables a sensitive and rapid analysis of gene products required for a normal (or transgenic) postembryonic phenotype. The example shown is a screen for genes involved in the subcellular localization of a fluorescently tagged protein. However, this protocol can be modified to identify genes affecting other postembryonic phenotypes of interest.

This method takes advantage of a candidate gene approach by using an RNAi library. Forward genetic screens usin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NGM Agar	Nematode growth medium	IPM Scientific, Inc	Can be prepared following NGM agar protocol25
M9 Medium			22mM KH2PO4, 42mM Na2HPO4, 86mM NaCl, 1 mM MgSO4
Agar-Agar	EMD Millipore	1.01614.1000	2% in water for NGM plates. 4% in water for microscope slide pads (autoclave initially and microwave to melt thereafter).
Bacto Peptone	BD Biosciences	211677	0.25%
IPTG	Research Products International Corp.	I56000-5.0	1 mM final concentration
carbenicillin	Research Products International Corp.	C46000-5.0	50 μg/ml working dilution
LB Broth Lennox	BD Biosciences	240230	20 g/liter
tetracycline	Sigma-Aldrich	268054	12.5 μg/ml working dilution
sodium hypochlorite	Any Supplier	5% household bleach	Use fresh bleach.
sodium hydroxide	Any Supplier	CAS 1310-73-2	5 N stock
M9 medium	Wormlab Recipe Book	http://130.15.90.245/wormlab_recipe_book.htm#Commonlab	26
levamisol	Sigma-Aldrich	31742	100 μM - 1 mM working dilution
sodium azide	Fisher Scientific	S227	10 mM in M9 working dilution
24-well plate	Greiner Bio-One	662160	VWR distributor
microscope slides	Any Supplier	75 x 25 x 1 mm
microscope cover slips	Any Supplier	22 x 22 mm No.1.5	Use the thickness recommended by the microscope manufacturer.
compound microscope	Carl Zeiss, Inc.	A1m	Use objectives and filters to match the needs of the experiment.
media pump	Manostat Varistaltic pump	Kate model #72-620-000	Use tubing and settings appropriate for the machine