Microinjection: Method of gene transfer
Microinjection is a direct type of gene transfer. It is also known as pronuclear microinjection. Microinjection is a very useful technique for transferring genetic materials into a living cell using a very thin needle. These needles can be glass micropipettes or metal microinjection needles. The transgene is in the form of plasmids, cosmids, phage, YACs, or PCR products. It can be circular or linear and need not be physically linked for injection. The main objective is generally a living cell but may also comprise intercellularly space. Microinjection is a quite mechanical process usually involving an inverted microscope.
Microinjection requires the direct mechanical introduction of DNA into the nucleus or cytoplasm using a glass microcapillary injection pipette. The protoplasts are immovable in low melting agar while working under a microscope, using a holding pipette and suction force.
For processes, such as cellular or pronuclear injection the target cell is positioned under the microscope and two micro-manipulators are required. One is holding the pipette and the other one holding a microcapillary needle usually between 0.5 and 5 micrometres in diameter (larger if injecting stem cells into an embryo)—which are used to penetrate the cell membrane and the nuclear envelope. In this manner, the process can be used to introduce a vector into a single cell. It can further be used in the cloning of organisms, in the study of cell biology, the study of viruses, and for treating male subfertility through intracytoplasmic sperm injection.
HISTORY OF MICROINJECTION:
In the early 20th century, the use of microinjection as a biological procedure introduce. Still in the 1970s it was not generally used. By the 1990s, its use had intensified significantly and is now treated as a common laboratory technique. Other laboratory techniques include vesicle fusion, electroporation, chemical transfection, and viral transduction for introducing a small substance into a small target. Marshall A. Barber suggested a new technique – the microinjection technique. He developed this technique initially to clone bacteria and to confirm the germ theory of Koch and Pasteur.
In the 1900s, it was first introduced with the injection of large macromolecules into cells. The method was later adapted to inject DNA, RNA, enzymes, proteins, metabolites, ions, and organelles into cells. In the development of microinjection techniques, Xenopus eggs, and animal embryos have been used largely because of their large size.
In this microinjection technique, a traditional compound microscope (around 200X magnification) or an inverted microscope (around 200x magnification), or a dissecting stereomicroscope (around 40-50x) is used. Under the microscope, an objective cell is positioned and the cell membrane and nuclear envelope are penetrated with the help of two micromanipulators. One micromanipulator holds the pipette and another one holds the microcapillary needle.
TYPES OF MICROINJECTION SYSTEMS:
These are the two basic types of microinjection systems-
- The first is called a constant flow system
- The second is called a pulsed flow system
Constant flow system:
In the constant flow system, a sample is delivered from a micropipette. The amount of sample injected is resolved by the duration for which the needle remains in the cell. It is relatively clumsy, simple, and inexpensive but outmoded. It requires a regulated pressure source, a capillary holder, and either a coarse or a fine micromanipulator.
Pulse flow system:
The pulsed flow system has greater control and firmness over the amount of substance or sample delivered. The most frequent settlement for intracytoplasmic sperm injection includes an Eppendorf “Femtojet” injector coupled with an Eppendorf “InjectMan”. Though the procedures involving other targets usually, take recognition of much less expensive equipment of similar capability. Because of its increased control over needle placement, and movement and increased precision over the volume of substance delivered. As a result, the pulsed flow technique causes less damage to the receiving cell than the constant flow technique.
However, the Eppendorf line, at least, has a complicated user interface and components of its particular system are usually much more costly than those necessary to create a constant flow system or than other pulsed flow injection systems.
Microinjection is an effective technique that plays an important part in the generation of genetically modified mouse and rat copies. Different types of microinjection systems can occur, the construction and configuration of which directly depends on the type of experiment.
Microinjection of DNA is the dominating technique popular with random integration of a transgene via the introduction of DNA into the pronucleus of a developing zygote. Following fertilization of a mouse egg, the male and female pronuclei survive and separated for some hours before they fuse to make the zygotic nucleus. So, this allows for the microinjection of the desired genes into the larger male pronucleus. Eggs that can endure the injections are transferred into the oviducts of a foster mother i.e., pseudopregnant female mice for the reproduction of the founder mouse, from which permanent transgenic lines can be well-established. The existence of transgenes in the offspring can be identified by PCR analysis or Southern blot hybridization.
Two kinds of DNA injections are included in the list-
- Plasmid vectors
- BAC Injections
- The number of DNA delivered per cell is not narrow and the delivery is precise, both of which raise the chance for integrative transformation.
- For a direct physical approach, this technique is host-range self-sufficient.
- Cells that are injected with the DNA material have tremendous regeneration potential.
- Despite pronuclear microinjection being popular, this method for transgenesis has some disadvantages such as a low success rate and mosaic founders.
ADVANTAGES OF MICROINJECTION:
- The amount of DNA delivered per cell is not finite by the techniques.
- It can be low toxicity.
- It can be optimized and it also improves the chances of integrative transformation.
- The distribution a correct, again increasing the chance for integrative transformation.
- The small structures can be injected containing only some of a few sales and with high regeneration potential.
- Therefore, it is a direct physical approach, it is host-range independent
DISADVANTAGES OF MICROINJECTION:
- The injection can cause damage that affects embryonic survival. As a result of quite a high fatality rate.
- Only one cell is aimed per injection.
- It requires specialized equipment as well as equipment users.
- Must have some knowledge about the technical skills to prevent cell damage.
To conclude, microinjection is a technique by which recombinant DNA can be easily injected into the nucleus of an animal as well as a human. Microinjection can be used to deliver antibodies targeted to a specific protein domain to evaluate the need for the protein for specific cell functions such as cell cycle progression, transcription of specific genes, or intracellular transport. It also helps in treating male subfertility. Overall, the introduction of microinjection is a boon for us.
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