Pronuclear injection is a laboratory method for creating transgenic animals, which are organisms that have had their genetic material altered. This technique is used to investigate what happens when a gene is overexpressed or expressed in an unusual way. By introducing a specific DNA sequence into a fertilized egg, scientists can produce animals, typically mice, that carry this new genetic information in their cells. This approach serves as a foundational tool in genetics and biomedical science, allowing researchers to explore the roles of specific genes and create models of human diseases.
Essential Concepts for Understanding Genetic Modification
To understand pronuclear injection, it is helpful to be familiar with some basic biological concepts. All living things are built using instructions encoded in a molecule called deoxyribonucleic acid, or DNA. Specific segments of this DNA are known as genes, which provide the blueprint for producing proteins and other functional molecules that determine an organism’s traits. The process by which the information in a gene is used is called gene expression.
The creation of a new animal begins with a fertilized egg, also called a zygote, which is a single cell formed when a sperm and egg unite. For a short period after fertilization, the genetic material from the sperm and the egg are not yet fused; they exist as two separate nuclei within the cell. These are called pronuclei, and it is into one of these that new DNA is injected. An animal that has had foreign DNA successfully added to its genome is known as a transgenic animal.
The Step-by-Step Pronuclear Injection Method
The process of pronuclear injection begins with the preparation of a DNA construct. This is the piece of genetic material, containing the gene of interest, that scientists intend to insert into the host animal’s genome. This DNA is isolated and purified before it can be used for injection. At the same time, fertilized eggs are collected from female animals, often mice, that have been mated. To increase the number of available eggs, the females may be treated with hormones to induce superovulation.
With the DNA construct and fertilized eggs prepared, the microinjection procedure can take place under a high-powered microscope. The fertilized egg is held in place by a small pipette, while a fine glass needle is used to inject the DNA solution directly into one of the two pronuclei. This is a delicate operation that requires precision, as the needle must pierce the cell membrane and the nuclear envelope without causing lethal damage to the single-celled embryo. The visibility of the pronuclei in mouse eggs makes them particularly suitable for this technique.
Creating and Identifying Transgenic Animals
After the microinjection is complete, the modified eggs are surgically transferred into a surrogate mother. This female has been made pseudopregnant, meaning she is hormonally prepared to support a pregnancy, typically by being mated with a vasectomized male. The surrogate mother then carries the embryos through gestation, which in mice lasts about 19 to 21 days, and gives birth to a litter of pups.
Not all of the resulting offspring will have successfully incorporated the injected DNA into their genome. For the new gene to be present in all the animal’s cells, it must integrate into the embryo’s own DNA at this single-cell stage. This integration happens at a random location within the genome, which means each resulting transgenic animal is unique. To determine which of the pups are transgenic, a small tissue sample is taken when they are a few weeks old and analyzed using Polymerase Chain Reaction (PCR) to detect the presence of the foreign DNA.
Scientific Goals of Pronuclear Injection
The purpose of creating transgenic animals through pronuclear injection is to study the function of genes. By introducing a new gene or a modified version of an existing one, researchers can observe the effects on the animal’s development, health, and behavior. This is useful for gain-of-function studies, where scientists investigate the consequences of a gene being overactive or expressed in tissues where it is not normally found. This can reveal information about the gene’s normal role in the body.
An application of this technology is the creation of animal models for human diseases. Scientists can introduce a gene known to be associated with a condition in humans into an animal like a mouse. This allows the animal to develop symptoms or pathologies similar to the human disease. These models are then used to study how the disease progresses and to test the safety and effectiveness of new drugs and therapies before they are considered for human trials. This technique has advanced the understanding of numerous genetic disorders and other complex diseases.