C. elegans Protocol Overview

C. elegans (Caenorhabditis elegans) is a widely used genetic model organism in developmental biology, neurobiology, aging, drug discovery, and toxicology. Below is an overview of common experimental protocols and workflows.

Protocol Types

Maintenance & Culturing

  • Strain Handling: Maintain worms on NGM (nematode growth medium) agar plates seeded with E. coli OP50.
  • Growth Conditions: Typically grown at 20°C; temperature shifts (15°C or 25°C) used for developmental timing or conditional mutants.
  • Life Stages: Embryo → L1 → L2 → L3 → L4 → Adult (~3 days at 20°C).

Synchronization of Worm Populations

  • L1 Synchronization (Starvation): Eggs are isolated via bleaching and hatched in M9 buffer without food to arrest at L1.
  • Bleach Protocol: Adult worms are lysed using bleach/NaOH to release embryos, which resist lysis and are used for timed experiments.

Mutagenesis

  • EMS Mutagenesis: Chemical mutagen ethyl methanesulfonate used to introduce random point mutations.
  • UV or ENU Alternatives: Occasionally used depending on mutagenesis goals.
  • Purpose: Forward genetic screens or mutant strain generation.

Genome Editing (CRISPR/Cas9)

  • Gene Knockout/Knock-in: Cas9 and sgRNA injected into the germline to induce targeted edits.
  • Repair Templates: ssODNs or plasmid donors used for HDR-based edits.
  • Applications: Functional genomics, humanized alleles, fluorescent tagging.

Phenotypic Screening

  • Developmental & Morphological Assays: Observe body shape, vulva morphology, growth rate, and lethality.
  • Behavioral Assays: Include locomotion, chemotaxis, thermotaxis, egg-laying, and feeding.
  • Lifespan & Healthspan: Used in aging studies; often combined with pharmacological interventions.

Drug Screening & Toxicology

  • Compound Administration: Via liquid culture, agar plates, or microfluidic devices.
  • Endpoints: Survival, reproduction, movement, oxidative stress response, gene expression.

RNAi by Feeding

  • Mechanism: Gene silencing via ingestion of E. coli expressing dsRNA.
  • System: Commonly performed in HT115 strain with L4440 vector.
  • Applications: Functional genomics, screening, pathway analysis.

Imaging & Fluorescent Reporters

  • Microscopy: Brightfield, DIC, and fluorescence imaging to observe transgenic reporters or labeled proteins.
  • Confocal Imaging: High-resolution subcellular localization or developmental analysis.
  • Live Imaging Tools: GFP, mCherry, GCaMP, and biosensors.

Molecular Biology & Omics

  • Sample Collection: Synchronized worms harvested for RNA, DNA, or protein extraction.
  • Applications: qPCR, RNA-seq, proteomics, metabolomics, ChIP.

Crossing & Genetics

  • Hermaphrodite/Male Crosses: Used for strain construction, mapping, or generating heterozygotes.
  • Balancer Chromosomes & Marker Strains: Essential for maintaining lethal or sterile alleles.

Optional Add-ons / Enhancements

  • Microinjection Services (e.g., transgene or CRISPR delivery)
  • Strain Banking and cryopreservation
  • Automated Worm Tracking and behavioral analysis
  • HTS Platforms for drug and RNAi screens
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