High Throughput Microinjections of Sea Urchin Zygotes

Summary

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

Abstract

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Introduction

Efficient and reproducible treatment delivery is one of the main methodological challenges for researchers. Several methods have been established to transiently deliver treatment solutions into the eggs, embryos, and cells. These methods include electroporation (based on a generating transient pores in the membrane using short electrical pulses)^1,2, lipofection (delivery through the fusion of treatment-containing liposomes with the membrane)¹, microparticle bombardment¹ (DNA is precipitated on the micron-sized metal particles that are then used to penetrate the cells at high velocity), and transduction (virus is used as a delivery vehi....

Protocol

1. Preparation of Protamine Sulfate (PS) Coated Dishes

1. Prepare 1% solution of protamine sulfate (PS) by adding 0.5 g of PS to 50 ml of deionized, distilled water (ddH₂O) in a 50 ml conical tube. Shake well at high speed on a bench shaker at room temperature for 1-2 hr to ensure complete dissolution of PS. This solution can be stored at 4 °C for at least 3 months (make sure to completely dissolve gel-like precipitate before each use)³.
2. Take a sleeve of 60 mm x 15 mm polystyrene Petri dishes and lay out both lids and bottoms on the bench.
3. Pour 1% PS solution in each dish (both bottoms and lids can be used) just ....

Results

GFP and mCherry reporter constructs were in vitro transcribed and microinjected into the newly fertilized eggs. Embryos were incubated at 15 °C for 24 hr (until the blastula stage) and imaged using Zeiss Observer Z1 microscope. Injection of reporter constructs did not lead to any developmental defects (Figure 6). For quantification of fluorescent signals, image acquisition was performed at low magnification (100X) to maximally capture fluorescent pixels (Figures 6D-F). Fluoresce.......

Discussion

Microinjection is a powerful technique for delivering various treatments such as DNA, mRNA, recombinant proteins, loss of function and gain of function reagents, dyes and their combinations into eggs, embryos, and cells of various organisms^1-7. However, several considerations should be kept in mind when designing a microinjection experiment.

It is critically important to consider the solubility of the delivered treatment and the injection volume. If the microinjected solution tends .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glass Pasteur pipettes	VWR	14673-043
Inverted microscope  Axiovert 40 °C	Zeiss	4109431007990000	Injection microscope
Microloader tips	Eppendorf	5242 956.003	Load injection solution
Nylon filter mesh 80 μm	Amazon.com	03-80-37	Filter eggs to get rid of debris
P20 or P200 Aerosol Barrier Pipette Tips	Fisher Scientific	02707432 or 02707430	Part of a mouth pipette
Parafilm	Fisher Scientific	13 374 12	Part of a mouth pipette
Polyethylene tubing	Intramedic	PE-160	Part of a mouth pipette
Protamine sulfate	MP Biomedicals, LLC	194729	Attach dejellied eggs to injection dishes
Sea urchins S. purpuratus	Pt. Loma Marine Invertebrate Lab	N/A
Sea water	any pet store	Instant Ocean
Sterile 60 mm x 15 mm Polystyrene Petri Dish	Fisher Scientific	0875713A	Injection dishes
Three-Axis Coarse Positioning Micromanipulator MMN-1	Narishige	9124	Manipulate injection needle
Three-Axis Joystick Type Oil Hydraulic Fine Micromanipulator MMO-202ND	Narishige	9212	Manipulate injection needle
Transfer pipettes	Fisher Scientific	13-711-9AM
Vertical  needle puller	Narishige	PC-10	Pull injection needles