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.
The technique of introducing new genetic material into the germline of mammals has been a major development in biotechnology over the last decades.
“Transgenic animals” are animals whose chromosomes contain stable, integrated copies of exogenous genes, additional copies of endogenous genes or gene constructs.
They are frequently created by two different techniques:
1) microinjection of DNA into the pronucleus of zygotes and 2) injection of embryonic stem cells into blastocysts.
In this Application Note, these techniques, as well as another technique for creating chimeras, the production of tetraploid mouse embryos, are discussed.
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.
Two major applications for microinjection in C. elegans are the production of transgenic worms (Mello et al. 1991) and the knockdown of genes by RNA interference (Fire et al. 1998). This Userguide describes the microinjection technique in C. elegans (devices: FemtoJet express/TransferMan NK2)
Microinjection of plasmid DNA and double-stranded RNA into Drosophila melanogaster embryos has been used to manipulate gene expression in many different ways. The best-known application of microinjection techniques is the generation of transgenic flies by P-element induced germline transformation. This Userguide describes the set-up for fly embryo injections which we established in our laboratory to generate transgenic fly lines and to inject double-stranded RNA for knockdown experiments.
In this Userguide microinjection is used to study mRNA transport and localization in stage I-II Xenopus oocytes. During oogenesis in various animal models, maternal mRNAs and proteins are localized to specific regions of the oocyte. These macromolecules are maternal determinants, and they provide the basis for patterning in the developing embryo.
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.
Germline transformation via microinjection of DNA into Drosophila melanogaster has been used to interfere with gene expression and study their function for many years. Recently, Bischof and co-workers established a new system to circumvent typical limitations by optimizing a phiC31-based integration system . This integration system demonstrates to be extremely efficient in Drosophila embryo microinjection. Furthermore, in combination with the ease-of-use plus versatility of this technique, a systematic high throughput screening of large cDNA sets and regulatory elements is now feasible. In this Userguide, a step-by-step protocol using the Eppendorf programmable microinjector FemtoJet and ready to use Femtotip II microcapillaries is presented for this application.