Microinjection: Principles, Steps, Applications, Advantages, and Disadvantages (Comprehensive Guide)

Table of Contents

• Introduction to Microinjection
• Principle of Microinjection
• Steps in Microinjection
• Applications of Microinjection
• Advantages of Microinjection
• Disadvantages of Microinjection

Introduction to Microinjection

• Microinjection is a physical method used for gene transfer by directly introducing DNA or other genetic materials into cells using a small glass needle or micropipette.
• This technique enables efficient transfer and integration of desired genes into the host cell's genome.
• It offers precise control over delivering materials, making it a potent tool in various research fields.
• Dr. Marshall A. Barber introduced DNA microinjection in the early 19th century, and it has since evolved with advancements in biomedical science.
• Applications include transgenics, animal cloning, human infertility treatment, genetic engineering, and genome editing.

Principle of Microinjection

• Microinjection operates on the principle of injecting genetic material directly into individual cells using a fine glass needle, termed a micropipette.
• Essential components include a micromanipulator for precise positioning and a microinjector to apply hydrostatic pressure.
• The procedure is conducted under a powerful microscope to ensure accuracy and visibility.
• Genetic material, typically DNA, is delivered into the cell by injecting a fluid through the micropipette.
• The small tip diameter of the micropipette and controlled movements via the micromanipulator enable precise delivery of genetic material into target cells.

Steps in Microinjection

• Preparation of micropipette: Glass micropipettes are formed by heating and stretching to create a fine tip, typically around 0.5 mm in diameter, resembling an injection needle.
• Microscopic environment: The entire procedure is conducted under a powerful microscope for precise handling and observation of the cells and micropipette.
• Cell preparation: Target cells are placed in a suitable container for microinjection.
• Holding pipette: A holding pipette nearby applies gentle suction to stabilize the target cell during the injection process.
• Micromanipulator setup: The micropipette, containing the desired materials, is mounted on a micromanipulator, enabling exact positioning and movement.
• Approach to cell: The micropipette is carefully positioned near the cell membrane, ready for insertion.
• Injection process: The micropipette is inserted into the cell membrane, either into the cytoplasm or nucleus, based on the target site for genetic material delivery.
• Material delivery: Using a microinjector, hydrostatic pressure is applied to release fluid containing genetic material into the cell.
• Withdrawal: Post-injection, the micropipette is slowly withdrawn from the cell to minimize cellular damage and membrane disruption.
• Cell viability: Proper technique and high-quality micropipettes are crucial to minimize cell death during the microinjection procedure.

Applications of Microinjection

Some major applications of microinjection are:

• Production of transgenic animals: Microinjection is extensively utilized to introduce foreign DNA into fertilized eggs, facilitating the study of gene function and the creation of disease models.
• Chimeric transgenic mice: Microinjection played a crucial role in producing the first chimeric transgenic mice, which are valuable for studying embryonic development, tissue transplantation, and cell lineage.
• In vitro fertilization (IVF): Microinjection is integral to intracytoplasmic sperm injection (ICSI) in IVF, enabling successful fertilization in cases of male infertility and leading to term pregnancies and healthy births by injecting defective sperm directly into oocytes.
• Somatic cell nuclear transfer (SCNT): Microinjection is employed in SCNT to create genetically identical organisms by transferring a somatic cell into an enucleated oocyte.
• Human embryo research: Microinjection is pivotal in various studies involving human embryos, enabling precise genetic manipulations and research advancements.
• Neuroscience applications: Microinjection is valuable in neuroscience for delivering proteins, peptides, and cDNA constructs into the cytosol of primary cultured human neurons, a task challenging with other gene transfer techniques.

Advantages of Microinjection

• Absence of selection markers: Microinjection eliminates the need for selection markers such as antibiotic-resistance genes, simplifying the process and bypassing additional steps to identify and isolate transformed cells.
• Precision in delivery: Microinjection allows precise control over the volume and timing of material delivery, a challenge with methods like electroporation or transfection.
• Easy identification of injected cells: Co-injection of marker dyes or fluorescently labeled proteins facilitates straightforward identification of injected cells.
• Reduced protein preparation: Microinjection requires less protein preparation compared to electroporation, which is beneficial for experiments involving less abundant or expensive proteins and peptides.
• Reduced cell stress: Microinjection imposes less stress on cells, leading to lower cell death rates compared to chemical transfection or viral infection methods.

Disadvantages of Microinjection

• Vehicle controls necessary: Microinjection requires vehicle controls to assess impacts on cell viability and ensure accurate experimental outcomes.
• Technical expertise required: Mastering microinjection and maintaining cell viability demands significant technical skill, posing a limitation.
• Preference for alternative techniques: Techniques such as transfection, infection, and electroporation are increasingly favored over microinjection for delivering impermeable materials into single cells.
• Labor-intensive and time-consuming: Manual microinjection is intensive in labor and time, limiting its practicality for large-scale or high-throughput experiments.
• Scalability limitations: Microinjection typically involves injecting a small subset of cells within a larger culture, restricting scalability.
• Inadequate for certain applications: Microinjection is not suitable for efficiently transferring genetic material into a large number of cells, as required for techniques like Western blotting or purification.
• Challenges with specific proteins: Direct microinjection of proteins such as membrane proteins or neurotransmitter receptors presents technical challenges due to their properties and cellular localization.