Embryo Microinjection Techniques for Efficient Site-Specific Mutagenesis in Culex quinquefasciatus

作者： Michelle Bui1, Ming Li1, Robyn R Raban1, Nannan Liu2, Omar S Akbari1,3 1Section of Cell and Developmental Biology,University of California, San Diego, 2Department of Entomology and Plant Patholog,Auburn University, 3Tata Institute for Genetics and Society,University of California, San Diego

简介：

Overview

This protocol outlines microinjection techniques for Culex quinquefasciatus embryos, optimized for CRISPR/Cas9 gene editing. It enables efficient generation of heritable germline mutations for genetic research and vector control technologies.

Key Study Components

Area of Science

• Genetic engineering
• Vector biology
• CRISPR/Cas9 technology

Background

• Culex quinquefasciatus is an understudied disease vector.
• Microinjection techniques are crucial for developing genetic control tools.
• Existing protocols for other insect species are well-established.
• This method accommodates the unique biological traits of Culex species.

Purpose of Study

• To develop a microinjection protocol for Culex quinquefasciatus.
• To facilitate gene function studies and genetic control technologies.
• To optimize survival and mutagenesis rates in injected embryos.

Methods Used

• Egg collection and raft separation techniques.
• Preparation of halocarbon oil and injection mixture.
• Microinjection of embryos using a micromanipulator.
• Screening for mutant phenotypes post-injection.

Main Results

• Achieved 64-82% embryo survival rate.
• Somatic mutagenesis rates ranged from 37-57%.
• Germline mutagenesis rates exceeded 61%.
• Multiplexing sgRNAs increased mutagenesis rates to 86%.

Conclusions

• The protocol successfully generates somatic and germline mutations.
• Viable homozygous stocks of mutants were maintained in the lab.
• Proper preparation of materials enhances the microinjection process.

Frequently Asked Questions