Whether it is DNA, RNA or protein that researchers want to insert into cells, microinjection is a simple yet powerful tool to do so. Unlike electroporation, which is an electrical procedure that can disrupt cell membranes and cause other physiological disruptions, microinjection allows the introduction of molecules into living cells with minimal risk of damage to cellular structures or function.
However, the microinjection process can be challenging. The injection process is prone to error, and errors can result in the failure to achieve desired effects. This is particularly true when injecting into tissues that are atypically vascularized, or that contain cell types that are difficult to identify. In addition, the high injection volume (pico to nanoliters) and low precision of the instrument can make it challenging to determine exactly how much solution is injected. This can lead to significant variation in results, which may not be detected or quantified with conventional techniques.
To address these challenges, we developed a system that automates the injection process, called the Autoinjector. The Autoinjector consists of a microscope camera for visualizing the injection area, a three-axis manipulator for position control of the micropipette, and a custom pressure regulation system enabling programmatic control of injection pressure. Using this system, we successfully injected human fetal brain slices with a precision of 300–500 m, and identified the location of individual neurons within each slice.
Injections are made with RNase-free bidistilled water, and solutions always contain a fluorescently labeled dye at 2-5 mg/ml (Alexa-coupled dextrans or Lucifer Yellow; Thermo Fisher Scientific) to visualize injected cells and their progeny. In vitro transcribed (ivt) poly-A mRNA for a green fluorescent protein (RFP) was used to mark cell nuclei.
The Autoinjector can be used to perform manual or programmed microinjections in a variety of different tissues and developmental models. To date, the system has been used to inject DNA into human fetal brains, zebrafish embryos, chick embryos and Caenorhabditis elegans. It is also capable of identifying and targeting neurons in human post-natal brain tissue, and can be adapted to target other cell types and layers in different tissues or developmental model organisms.
The Autoinjector can also be used to deliver a library of mRNAs or DNA into a specific cell population, or to perform CRISPR/Cas9-mediated genetic disruption in specific cells. For these applications, a mRNA- or DNA-containing microfluid is placed into the open end of a micropipette and inserted into each cell trapping channel in a cell-trapping chip. Each micropipette is then programmed to dispense a specific amount of the molecule into each cell trapped in the chip. RNA-injections have been shown to be an efficient method to deliver mRNA pools for combinatorial analysis of gene expression during brain development 5-7. EdU incorporation 24 h after the microinjection was used to evaluate cell viability and to assess progression through the cell cycle. Compared to cells in an uninjected organotypic slice, injected cells showed significantly more EdU incorporation, indicating that the Autoinjector can accurately target a large number of neurons within a complex tissue. micro injection