Microinjection is one of the core methods to introduce DNA and other non-permeable molecules into cells. It allows direct access to the two main intracellular compartments, the nucleus and the cytoplasm. Furthermore, single cells can be used to study complex cellular processes, structure and function in vivo. However, sample preparation and handling is crucial for achieving successful microinjection and has to be done with great care. This application mainly focuses on microinjection into adherent cells. In addition, useful general information about sample preparation is presented.

Microinjection techniques are widely applied in developmental biology for the analysis of early developmental processes such as gastrulation, neural induction and patterning or organogenesis. Microinjection experiments into vertebrate embryos (e.g. mouse, frog, fish) allow to generate transgenic animals by injection of DNA; to interfere with specific developmental processes by DNA, RNA or morpholino oligo injection, or to follow the fate of individual cells by the injection of fluorescent lineage tracer dyes.

Microinjection is one of the core methods to introduce foreign DNA and other non-permeable molecules into cells. Nuclear injection of plasmid DNA enables rapid expression of proteins in specific cells within a population. The major advantage of this approach over other transient transfection methods is the rapid burst of expression that follows. In this article, we use this approach to introduce function-blocking mutants of proteins, inhibitory antibodies and cell impermeable chemical inhibitors to inhibit specific functions within the mammalian protein secretory pathway.

In this application note is described how the microinjection procedure into hydra embryos is performed with the aid of an Eppendorf Micromanipulator 5171, CellTram vario and a Zeiss Axiovert 100 inverted microscope.

Microinjection is one of the core methods to introduce DNA and other non-permeable molecules into cells. It allows single cell investigation to study complex cellular processes, structure and function in vivo. However, the first steps to micromanipulation are crucial for achieving successful microinjection and have to be done with great care. These steps consist of the set-up process of the workstation, including angle adjustment of the manipulator and fine adjustment and focussing of the capillary.

Microinjection techniques are widely applied in developmental biology for the analysis of early developmental processes such as gastrulation, neural induction and patterning or organogenesis. Microinjection experiments into vertebrate embryos (e.g. mouse, frog, fish) allow to generate transgenic animals by injection of DNA, to interfere with specific developmental processes (DNA, RNA, morpholino-injection), or to follow the fate of individual cells by the injection of fluorescent lineage tracer dyes.

In this application a robotic injection system consisting of the Eppendorf InjectMan NI2 and FemtoJet Express, coupled with COPAS sorting, is described as an extremely powerful tool for a high throughput platform for the in vivo study of diseases as tuberculosis that benefit from the use of whole vertebrate organism as zebrafish, even facilitating a high biosafety-level study.

In this application note we describe the set-up and workflow of an automatic injection system consisting in main parts of the Eppendorf InjectMan® NI 2, the FemtoJet® express microinjector and a motorized stage (Märzhäuser), controlled using a serial interface connected to a Linux computer with macro programs written in python which enable subsequent high-throughput compound-screening.