Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Negin P. Martin; Page Myers; Eugenia Goulding; Shih-Heng Chen; Mitzie Walker; Thomas M. Porter; Lucas Van Gorder; Amanda Mathew; Artiom Gruzdev; Erica Scappini; Charles Romeo

doi:10.3791/58327

Entrega de Gene lentivirales asistida por láser ratón huevos fertilizados

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Biology

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Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Entrega de Gene lentivirales asistida por láser ratón huevos fertilizados

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06:03 min

November 1, 2018

DOI: 10.3791/58327-v

Negin P. Martin*¹, Page Myers*², Eugenia Goulding¹, Shih-Heng Chen¹, Mitzie Walker¹, Thomas M. Porter¹, Lucas Van Gorder¹, Amanda Mathew¹, Artiom Gruzdev³, Erica Scappini⁴, Charles Romeo¹

¹Neurobiology Laboratory,National Institute of Environmental Health Sciences, ²Comparative Medicine Branch,National Institute of Environmental Health Sciences, ³Reproductive and Developmental Biology Laboratory,National Institute of Environmental Health Sciences, ⁴Signal Transduction Laboratory,National Institute of Environmental Health Sciences

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Overview

This study addresses gene delivery in mouse fertilized eggs by laser perforation of the zona pellucida, allowing for transduction with lentiviral vectors. The protocol demonstrates a method for creating transgenic mice with minimal specialized skills required, which could have applications in other species.

Key Study Components

Research Area

Genetics
Developmental biology
Gene delivery methods

Background

Zona pellucida as a barrier in early embryonic development
Challenges in gene delivery to embryonic cells
Importance of developing transgenic models for research

Methods Used

Laser-assisted perforation technique
Mouse embryos as a biological model
Lentiviral gene delivery system

Main Results

70% of healthy embryos develop into blastocysts; 47% of transduced embryos achieve the same
85% of transduced embryos express the gene of interest
Technique supports rapid development of transgenic animal models

Conclusions

The method effectively facilitates stable gene delivery in mouse embryos
Significant implications for in vivo studies related to genetic diseases

Frequently Asked Questions

What is the zona pellucida?

The zona pellucida is a glycoprotein matrix that surrounds mouse fertilized eggs and early-stage embryos, acting as a barrier to gene delivery.

Why use laser perforation?

Laser perforation allows for precise and rapid gene delivery to fertilized eggs without requiring specialized injection techniques.

What is the success rate of this technique?

About 47% of laser-perforated transduced embryos develop into blastocysts, with 85% expressing the transgene.

Can this method be applied to other species?

Yes, the technique may be applicable to the fertilized eggs of other species for gene delivery.

What safety precautions are necessary?

It is crucial to follow institutional guidelines for handling lentiviruses, including wearing personal protective equipment and decontaminating waste.

What are potential applications of this research?

The study could aid in the rapid development of transgenic animal models for gene function studies and disease research.

How does lentiviral transduction work?

Lentivirus integrates into the host genome, allowing for stable expression of the gene of interest but carries a risk of insertional mutations.

Ratón huevos fertilizados y embriones de etapa temprana están protegidos por la zona pelúcida, una matriz de la glicoproteína que forma una barrera contra la entrega del gene. Este artículo describe un protocolo para perforar la zona con un láser para transducir las células embrionarias con vectores lentivirales y crear ratones transgénicos.

Este es un método alternativo y fácil de usar para entregar genes de interés a los huevos de ratón fertilizados a través de la perforación láser de la zona pellucida y la entrega de genes lentivirales. La perforación asistida por láser del procedimiento de donante de ratón también puede aplicarse a los óvulos fertilizados de otras especies para facilitar la entrada de otros tipos de virus o reactivos de transfección. La principal ventaja de esta técnica es que no requiere habilidades especializadas para la micromanipulación y microinyección de los huevos de ratón fertilizados.

Demostrar el procedimiento de aislamiento de óvulos de ratón fertilizado será Page Myers, un científico de investigación de la rama de Medicina Comparada en el NIEHS. 16 a 24 horas después del apareamiento, aislar los ovarios y ovucetos de ratones hembra con tapones vaginales, de acuerdo con los protocolos estándar. Cuando se hayan recogido todos los tejidos, desgarra la ampolla de cada ovario y libera los óvulos fertilizados, rodeados de células cumulous, y transfiere los huevos en células cumulous a un plato de 35 milímetros que contiene una x hialuronidasa en medio M2 fresco, para una incubación de tres a cinco minutos a temperatura ambiente.

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