Microinjection is the use of a glassmicropipette to inject a liquid substance at amicroscopic or borderlinemacroscopic level. The target is often a living cell but may also include intercellular space. Microinjection is a simple mechanical process usually involving aninverted microscope with amagnification power of around 200x (though sometimes it is performed using a dissectingstereo microscope at 40–50x or a traditionalcompound upright microscope at similar power to an inverted model).
For processes such as cellular orpronuclear injection the target cell is positioned under the microscope and twomicromanipulators—one holding the pipette and one holding a microcapillary needle usually between 0.5 and 5 μm in diameter (larger if injecting stem cells into an embryo)—are used to penetrate thecell membrane and/or thenuclear envelope.[1] In this way the process can be used to introduce avector into a single cell. Microinjection can also be used in thecloning of organisms, in the study of cell biology and viruses, and for treating malesubfertility throughintracytoplasmic sperm injection (ICSI,/ˈɪksi/IK-see).
The use of microinjection as a biological procedure began in the early twentieth century, although even through the 1970s it was not commonly used. By the 1990s, its use had escalated significantly and it is now considered a common laboratory technique, along withvesicle fusion,electroporation,chemical transfection, andviral transduction, for introducing a small amount of a substance into a small target.[2]
There are two basic types of microinjection systems. The first is called aconstant flow system and the second is called apulsed flow system. In a constant flow system, which is relatively simple and inexpensive though clumsy and outdated, a constant flow of a sample is delivered from amicropipette and the amount of the sample which is injected is determined by how long the needle remains in the cell. This system typically requires a regulated pressure source, a capillary holder, and either a coarse or a fine micromanipulator. A pulsed flow system, however, allows for greater control and consistency over the amount of sample injected: the most common arrangement forintracytoplasmic sperm injection includes anEppendorf "Femtojet" injector coupled with an Eppendorf "InjectMan", though procedures involving other targets usually take advantage of much less expensive equipment of similar capability. Because of its increased control over needle placement and movement and in addition to the increased precision over the volume of substance delivered, the pulsed flow technique usually results in less damage to the receiving cell than the constant flow technique. However, the Eppendorf line, at least, has a complexuser interface and its particular system components are usually much more expensive than those necessary to create a constant flow system or than other pulsed flow injection systems.[3]
Pronuclear injection is a technique used to createtransgenic organisms by injecting genetic material into the nucleus of a fertilizedoocyte. This technique is commonly used to study the role of genes using mouse animal models.
The pronuclear injection of mouse sperm is one of the two most common methods for producing transgenic animals (along with the genetic engineering of embryonicstem cells).[4] In order for pronuclear injection to be successful, the genetic material (typically linearDNA) must be injected while the genetic material from the oocyte and sperm are separate (i.e., thepronuclear phase).[5] In order to obtain these oocytes, mice are commonlysuperovulated usinggonadotrophins.[6] Onceplugging has occurred,oocytes are harvested from the mouse and injected with the genetic material. The oocyte is then implanted in theoviduct of apseudopregnantanimal.[5] While efficiency varies, 10-40% of mice born from these implanted oocytes may contain the injectedconstruct.[6] Transgenic mice can then be bred to create transgenic lines.