Microinjection Technique And Applications: Pronuclear, Zebrafish and Other Examples
| April 11, 2023
Microinjection has major advancements since its invention a century ago. In the following, we will introduce the concept and components of a microinjection system, and illustrate some common experimental applications of microinjection, including Pronuclear and zebrafish microinjection.
The Concept Of Microinjection Technique
The microinjection technique, which was invented by the bacteriologist Dr. Marshall Barber from the University of Kansas, has existed over a century till now. In 1902, Dr. Barber used this method to separate the bacterial cells, and at the same time developed the first set of microinjection instruments. In 1919, Dr. Frederick Pratt and Dr. John Elsenberger used the nanoliter microinjection pump for the first time for partial stimulation of the skeletal muscle fiber. As the technique continued to grow and develop, there had been 5 groundbreaking projects related to microinjection that were awarded the Nobel Prize in Physiology or Medicine, including the embryonic induction theory in 1935, the patch clamp technique in 1991, the knockout mice experiment in 2007, IVF in 2010, the genetically modified and cloned animals in 2012. Currently, genetic engineering technology is rapidly developing. Applications such as the construction of genetically modified and cloned animals, and the studies on gene and cell development are becoming more prevalent.
With a microinjection system, the super small and thin microinjection glass capillary needle (also known as glass electrode) is used for implanting exogenous materials (DNA or RNA or cell) into the cells, leading to the occurrence of chimerism between exogenous materials and genomes in order to integrate the exogenous materials into the genome. The subjects and sites chosen for injection vary based on the goal, needs and design of the experiments.
Components of Microinjection System
One must have a set of highly calibrated RWD Microinjection system to carry out a highly precise experiment. First of all, a micropipette puller is needed for pulling out the super small and thin glass micropipette with a tip diameter requirement down to 1-30 um. Next, a highly accurate injection pump is needed for injection. One can choose to use or not to use the non-pressurized injection pump depending on the requirements of the experiment. Apart from that, a micromanipulator is needed for maintaining the stability of the injection pump. After all, it is impossible for human hands to manipulate the pump without drifting. Last but not least, a good microscope is needed for ensuring the accuracy of the injection position. Indeed, these instruments should be placed on the shock-resistant base to prevent any negative effects caused by drifting during the microinjection experiment.
Common Experimental Applications of Microinjection
1. Microinjection of Nematodes Caenorhabditis elegans (C. elegans) is an important model organism used for studying animal genetics, ontogeny, and ethology. It can help us to study the mechanism of action from the molecular and cellular level to system biological level in regard to the corresponding life cycle. Microinjection is the core technique in the C. elegans research paradigm. It is widely used in studying gene expression, gene function and genetic interactions inside the body of C. elegans.
2. Zebrafish Microinjection In microinjection experiments, zebrafish is a model organism which has been widely used, since it grows faster than any other mammals. Thus, it is easier to obtain its embryos. Substances such as DNA and RNA are introduced into the developing embryo through microinjection techniques, providing researchers with the fastest and most reliable means to the preparation of genetically modified or mutated zebrafish species.
3.Xenopus MicroInjection Xenopus embryos are commonly used in researches such as studying the effects of the nervous system on cell level and investigating how the nerve development forms the correct links. These embryos have big and identifiable blastomeres. Being important models for the research of embryo development, they have other advantages such as high tolerance to different types of surgical intervention and external breeding. Therefore, Xenopus embryos have become significant and unique resources for the research of early embryo development and cell biology.
4.Microinjection of Insects (fruit flies/ mosquitoes etc.) Biologists are in favor of using insects like fruit flies and mosquitoes as they have advantages such as small size, fast growth and easy operation. They are widely used in research fields including genetics, evolutionary biology and developmental biology, especially the creation of different genetically modified species.
5.Pronuclear Microinjection Pronuclear microinjection introduces exogenous DNA parts into the nuclei of fertilised eggs to integrate the DNA into the existing genome under the assistance of a microinjection operation system. This kind of integration creates a new copy of the genome for every single cell during the birth of animals. After that, the embryo will be transferred to the surrogate body by using embryo transfer technology for growth and development. Such technique is very convenient, time-saving and cost-effective for experiments. Therefore, it is one of the most effective and conventional ways to create genetically modified mice.
6.Intracytoplasmic Sperm Microinjection Intracytoplasmic Sperm Injection (ICSI) is an advanced form of IVF. A single sperm cell is extracted and then directly injected into the parent cell of an egg cell to carry out fertilisation. It is honoured as one of the most important breakthroughs in the reproductive biomedical research field. The applications of this technique can be extended to other species, usually including cattle, sheep, horses, rabbits, pigs and mice.
7.Microinjection of Embryonic Stem Cells Embryonic stem cells (ESC) are cells that can be divided to form any cell types from the three embryonic layers, but they cannot form other associative and supportive tissues such as placenta and umbilical cord. These stem cells have multifaceted features. The ESC microinjection technique has great influence over the research and studies of gene function, changes in gene expression and gene regulation.