Generating Transgenics and Knockouts in Strongyloides Species by Microinjection
October 7th, 2021
The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S. stercoralis and S. ratti.
Overview
This protocol outlines the use of intragonadal microinjection for introducing transgenes and CRISPR components into the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti. This method is crucial for generating knockouts and transgenics to study the development and parasitism of these worms.
Key Study Components
Area of Science
- Parasitology
- Genetics
- Microinjection Techniques
Background
- Strongyloides stercoralis and Strongyloides ratti are parasitic nematodes.
- Understanding their biology is essential for controlling infections.
- Current methods for genetic manipulation are limited.
- This protocol provides a reliable technique for genetic studies.
Purpose of Study
- To demonstrate a method for introducing genetic material into nematodes.
- To facilitate the study of gene function in parasitic behavior.
- To advance the understanding of nematode biology through genetic tools.
Methods Used
- Intragonadal microinjection of DNA constructs.
- Preparation of agarose pads for microinjection.
- Use of a Baermann holder for nematode handling.
- CRISPR/Cas9-mediated mutagenesis and RNA interference techniques.
Main Results
- Successful generation of knockouts and transgenics in S. stercoralis and S. ratti.
- Demonstrated effectiveness of the microinjection technique.
- Provided insights into the developmental processes of these nematodes.
- Established a foundation for future genetic studies in parasitic nematodes.
Conclusions
- The intragonadal microinjection method is effective for genetic manipulation.
- This technique opens new avenues for research on parasitic nematodes.
- Further studies can enhance our understanding of host-parasite interactions.
Frequently Asked Questions
Executive Industry Relevance
Genetic manipulation of parasitic nematodes enables target validation and mechanistic de-risking in antiparasitic drug discovery. Intragonadal microinjection provides a reliable method to generate transgenics and knockouts in Strongyloides species, supporting phenotypic screening and assay development for host-parasite interaction studies. This technique advances preclinical model relevance by facilitating functional gene analysis in pathogenic nematodes.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of gene function in parasitic development and host invasion mechanisms.
- Operational Value: Supports target confirmation through phenotypic analysis of knockout and transgenic lines.
Screening & Assay Development
- Scientific Value: Generates transgenic reporter lines for quantifying gene expression in live parasites.
- Operational Value: Enables standardized preparation of microinjection-ready nematodes for reproducible assay inputs.
Translational & Preclinical Research
- Scientific Value: Facilitates study of anthelmintic resistance mechanisms through targeted gene disruption.
- Operational Value: Provides disease-relevant systems for evaluating compound effects on genetically defined parasites.
Pipeline & Workflow Integration
The method supports early discovery workflows by enabling target validation through genetic perturbation, with outputs feeding into phenotypic screening and lead identification stages.
- Discovery Biology: Permits hypothesis testing via gonad-specific delivery of CRISPR/Cas9 or transgene constructs.
- Screening: Produces transgenic larvae with detectable reporters for fluorescence-based assay readouts.
- Analytics: Yields quantitative expression patterns to assess genetic modification efficiency and phenotypic impact.
- Translational Research: Connects genetic findings to preclinical models of host-parasite interaction.
- Enterprise Reuse: Establishes a scalable platform for genetic manipulation across Strongyloides species.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in target role through direct genetic manipulation in pathogenic nematodes.
- Operational Value: Standardized microinjection protocol ensures reproducibility across laboratories.
- Strategic Value: Reduces biological risk in target selection by confirming gene function in vivo.
- Portfolio Impact: Enables data-driven prioritization of antiparasitic candidates based on validated targets.
Implementation Considerations
- Expertise in nematode dissection and microinjection techniques.
- Apparatus for agarose pad preparation and Baermann-based worm isolation.
- Fluorescence microscopy for transgenic larva identification and tracking.
- Standardized protocols for worm recovery and post-injection recovery plating.
- Skill in gonad targeting and needle alignment for efficient DNA delivery.
Biopharma FAQs
Chapters in this video
0:05
Introduction
0:37
Preparation of Microinjection Slides
1:19
Collection of Young Adult Strongyloides for Microinjection
3:19
Preparing the Microscope and Breaking the Needle
5:27
Microinjecting Strongyloides
7:22
Collecting and Screening F1 Larvae to Recover Transgenics/Knockouts
8:31
Results: Generating Transgenics and Knockouts in Strongyloides Species Using Intragonadal Microinjection
9:04
Conclusion
