Microinjection

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Microinjection of a fluorescent dye into Ciona intestinalis eggs positioned in a microwell array.

Microinjection is the use of a glass micropipette to inject a liquid substance at a microscopic or borderline macroscopic level. The target is often a living cell but may also include intercellular space. Microinjection is a simple mechanical process usually involving an inverted microscope with a magnification power of around 200x (though sometimes it is performed using a dissecting stereo microscope at 40–50x or a traditional compound upright microscope at similar power to an inverted model).

Contents

For processes such as cellular or pronuclear injection the target cell is positioned under the microscope and two micromanipulators—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 the cell membrane and/or the nuclear envelope. [1] In this way the process can be used to introduce a vector into a single cell. Microinjection can also be used in the cloning of organisms, in the study of cell biology and viruses, and for treating male subfertility through intracytoplasmic sperm injection (ICSI, /ˈɪksi/ IK-see).

History

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 with vesicle fusion, electroporation, chemical transfection, and viral transduction, for introducing a small amount of a substance into a small target. [2]

Basic types

There are two basic types of microinjection systems. The first is called a constant flow system and the second is called a pulsed 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 In a constant flow system, which is relatively simple and inexpensive though clumsy and outdated, a constant flow of a sample is delivered from a micropipette and the amount of the sample which is injected is determined by how long the needle remains in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 for intracytoplasmic sperm injection includes an Eppendorf "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 complex user 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

Diagram of the intracytoplasmic sperm injection of a human egg. Micromanipulator on the left holds egg in position while microinjector on the right delivers a single sperm cell. Microinjection of a human egg.svg
Diagram of the intracytoplasmic sperm injection of a human egg. Micromanipulator on the left holds egg in position while microinjector on the right delivers a single sperm cell.

Pronuclear injection is a technique used to create transgenic organisms by injecting genetic material into the nucleus of a fertilized oocyte. This technique is commonly used to study the role of genes using mouse animal models.

Pronuclear injection in mice

The pronuclear injection of mouse sperm is one of the two most common methods for producing transgenic animals (along with the genetic engineering of embryonic stem cells). [4] In order for pronuclear injection to be successful, the genetic material (typically linear DNA) must be injected while the genetic material from the oocyte and sperm are separate (i.e., the pronuclear phase). [5] In order to obtain these oocytes, mice are commonly superovulated using gonadotrophins. [6] Once plugging has occurred, oocytes are harvested from the mouse and injected with the genetic material. The oocyte is then implanted in the oviduct of a pseudopregnant animal. [5] While efficiency varies, 10-40% of mice born from these implanted oocytes may contain the injected construct. [6] Transgenic mice can then be bred to create transgenic lines.

See also

Related Research Articles

Pipette Liquid-transferring laboratory tool

A pipette is a laboratory tool commonly used in chemistry, biology and medicine to transport a measured volume of liquid, often as a media dispenser. Pipettes come in several designs for various purposes with differing levels of accuracy and precision, from single piece glass pipettes to more complex adjustable or electronic pipettes. Many pipette types work by creating a partial vacuum above the liquid-holding chamber and selectively releasing this vacuum to draw up and dispense liquid. Measurement accuracy varies greatly depending on the style.

Intracytoplasmic sperm injection in vitro fertilization procedure

Intracytoplasmic sperm injection is an in vitro fertilization (IVF) procedure in which a single sperm cell is injected directly into the cytoplasm of an egg. This technique is used in order to prepare the gametes for the obtention of embryos that may be transferred to a maternal uterus. With this method acrosome reaction is skipped.

Acrosome reaction The discharge, by sperm, of a single, anterior secretory granule following the sperms attachment to the zona pellucida surrounding the oocyte. The process begins with the fusion of the outer acrosomal membrane with the sperm plasma membrane and ends

During fertilization, a sperm must first fuse with the plasma membrane and then penetrate the female egg cell in order to fertilize it. Fusing to the egg cell usually causes little problem, whereas penetrating through the egg's hard shell or extracellular matrix can present more of a problem to the sperm. Therefore, sperm cells go through a process known as the acrosome reaction which is the reaction that occurs in the acrosome of the sperm as it approaches the egg. The acrosome is a cap-like structure over the anterior half of the sperm's head.

Nuclear transfer

Nuclear transfer is a form of cloning. The steps involve removing the DNA from an oocyte, and injecting the nucleus which contains the DNA to be cloned. In rare instances, the newly constructed cell will divide normally, replicating the new DNA while remaining in a pluripotent state. If the cloned cells are placed in the uterus of a female mammal, a cloned organism develops to term in rare instances. This is how Dolly the Sheep and many other species were cloned. Cows are commonly cloned to select those that have the best milk production. On 24 January 2018, two monkey clones were reported to have been created with the technique for the first time.

Pronucleus The nucleus of either the ovum or the spermatozoon following fertilization. Thus, in the fertilized ovum, there are two pronuclei, one originating from the ovum, the other from the spermatozoon that brought about fertilization; they approach each oth

A pronucleus is the nucleus of a sperm or an egg cell during the process of fertilization. The sperm cell becomes a pronucleus after the sperm enters the ovum, but before the genetic material of the sperm and egg fuse. Contrary to the sperm cell, the egg cell has a pronucleus once it becomes haploid, and not when the sperm cell arrives. Sperm and egg cells are haploid, meaning they carry half the number of chromosomes of somatic cells, so in humans, haploid cells have 23 chromosomes, while somatic cells have 46 chromosomes. The male and female pronuclei don't fuse, although their genetic material does. Instead, their membranes dissolve, leaving no barriers between the male and female chromosomes. Their chromosomes can then combine and become part of a single diploid nucleus in the resulting embryo, containing a full set of chromosomes.

Polly and Molly, two ewes, were the first mammals to have been successfully cloned from an adult somatic cell and to be transgenic animals at the same time. This is not to be confused with Dolly the Sheep, the first animal to be successfully cloned from an adult somatic cell where there was no genetic modification carried out on the adult donor nucleus. Polly and Molly, like Dolly the Sheep, were cloned at the Roslin Institute in Edinburgh, Scotland.

A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques from one organism to another. The introduction of a transgene (catransgenesis") has the potential to change the phenotype of an organism. Transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporated into the organism's germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that particular gene.

Megan and Morag, two domestic sheep, were the first mammals to have been successfully cloned from differentiated cells. They are not to be confused with Dolly the sheep which was the first animal to be successfully cloned from an adult somatic cell or Polly the sheep which was the first cloned and transgenic animal. Megan and Morag, like Dolly and Polly, were cloned at the Roslin Institute in Edinburgh, Scotland in 1995.

Ralph L. Brinster American geneticist

Ralph Lawrence Brinster is an American geneticist and Richard King Mellon Professor of Reproductive Physiology at the School of Veterinary Medicine, University of Pennsylvania.

Sperm-mediated gene transfer (SMGT) is a transgenic technique that transfers genes based on the ability of sperm cells to spontaneously bind to and internalize exogenous DNA and transport it into an oocyte during fertilization to produce genetically modified animals.1 Exogenous DNA refers to DNA that originates outside of the organism. Transgenic animals have been obtained using SMGT, but the efficiency of this technique is low. Low efficiency is mainly due to low uptake of exogenous DNA by the spermatozoa, reducing the chances of fertilizing the oocytes with transfected spermatozoa.2 In order to successfully produce transgenic animals by SMGT, the spermatozoa must attach the exogenous DNA into the head and these transfected spermatozoa must maintain their functionality to fertilize the oocyte.2 Genetically modified animals produced by SMGT are useful for research in biomedical, agricultural, and veterinary fields of study. SMGT could also be useful in generating animals as models for human diseases or lead to future discoveries relating to human gene therapy.

Micromanipulator

A micromanipulator is a device which is used to physically interact with a sample under a microscope, where a level of precision of movement is necessary that cannot be achieved by the unaided human hand. It may typically consist of an input joystick, a mechanism for reducing the range of movement and an output section with the means of holding a microtool to hold, inject, cut or otherwise manipulate the object as required. The mechanism for reducing the movement usually requires the movement to be free of backlash. This is achieved by the use of kinematic constraints to allow each part of the mechanism to move only in one or more chosen degrees of freedom, which achieves a high precision and repeatability of movement, usually at the expense of some absolute accuracy.

Genetically modified mouse

A genetically modified mouse is a mouse that has had its genome altered through the use of genetic engineering techniques. Genetically modified mice are commonly used for research or as animal models of human diseases, and are also used for research on genes.

Knockout rat genetically engineered rat with a single gene turned off through a targeted mutation used for academic and pharmaceutical research

A knockout rat is a genetically engineered rat with a single gene turned off through a targeted mutation used for academic and pharmaceutical research. Knockout rats can mimic human diseases and are important tools for studying gene function and for drug discovery and development. The production of knockout rats was not economically or technically feasible until 2008.

The Genetics & IVF Institute (GIVF) is an international provider of infertility and genetics services and products, and also engages in biomedical research in these fields. The Institute was founded in 1984 by Dr. Joseph D. Schulman and associates. GIVF headquarters are in Fairfax, VA, US, and its facilities include locations in Pennsylvania, Minnesota, California, and Texas in the United States, as well as in China, Mexico, and several other countries.

Teratospermia or teratozoospermia is a condition characterized by the presence of sperm with abnormal morphology that affects fertility in males.

Oocyteactivation is a series of processes that occur in the oocyte during fertilization.

Nanoinjection is the process of using a microscopic lance and electrical forces to deliver DNA to a cell. It is claimed to be more effective than microinjection because the lance used is ten times smaller than a micropipette and the method uses no fluid. The nanoinjector mechanism is operated while submerged in a pH buffered solution. Then, a positive electrical charge is applied to the lance, which accumulates negatively charged DNA on its surface. The nanoinjector mechanism then penetrates the zygotic membranes, and a negative charge is applied to the lance, releasing the accumulated DNA within the cell. The lance is required to maintain a constant elevation on both entry and exit of the cell.

Breast cancer metastatic mouse models are experimental approaches in which mice are genetically manipulated to develop a mammary tumor leading to distant focal lesions of mammary epithelium created by metastasis. Mammary cancers in mice can be caused by genetic mutations that have been identified in human cancer. This means models can be generated based upon molecular lesions consistent with the human disease.

Globozoospermia male infertility characterized by round-headed spermatozoa lacking an acrosome

Globozoospermia is a rare and severe form of monomorphic teratozoospermia. This means that the spermatozoa show the same abnormality, and over 85% of spermatozoa in sperm have this abnormality. Globozoospermia is responsible for less than 0.1% of male infertility. It is characterised by round-headed spermatozoa without acrosomes, an abnormal nuclear membrane and midpiece defects. Affected males therefore suffer from either reduced fertility or infertility. Studies suggest that globozoospermia can be either total or partial however it is unclear whether these two forms are variations on the same syndrome, or actually different syndromes.

The intracytoplasmic morphologically selected sperm injection (IMSI) is a laboratory technique used for In Vitro Fertilisation treatments. High quality sperms are injected into the egg for fertilization, it is an advanced version of ICSI. A high powered microscope is used to pick out and the best sperm cells which are then used in a traditional ICSI protocol. This allows the sperm to be examined in greater detail, including the nucleus which contains the sperm's genetic material. Use of this method has resulted in higher pregnancy and delivery rates and lower abortion rates. IMSI is a good choice for anyone who has failed IVF cycles in the past, and for couples who have a component of male infertility.

References

  1. ↑ David B. Burr; Matthew R. Allen (11 June 2013). Basic and Applied Bone Biology. Academic. p. 157. ISBN   978-0-12-391459-0 . Retrieved 15 July 2013.
  2. ↑ Juan Carlos Lacal; Rosario Perona; James Feramisco (11 June 1999). Microinjection. Springer. p. 9. ISBN   978-3-7643-6019-1 . Retrieved 13 July 2013.
  3. ↑ Robert D. Goldman; David L. Spector (1 January 2005). Live Cell Imaging: A Laboratory Manual. CSHL. p. 54. ISBN   978-0-87969-683-2 . Retrieved 15 July 2013.
  4. ↑ Heinz Peter Nasheuer (2010). Genome Stability and Human Diseases. Springer. p. 328. ISBN   978-90-481-3471-7 . Retrieved 15 July 2013.
  5. 1 2 Mullin, Ann. "Pronuclear Injection". Tulane University.
  6. 1 2 "Pronuclear Injection". UC San Diego . Retrieved 6 December 2019.
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