Wednesday 17th April 2024
Durbar Marg, Kathmandu

Introducing foreign DNA into host cells is an essential step in many biological experiments. There are several chemical and physical transformation techniques available, but microinjection is the most direct method. Using very small bore glass needles with an outer diameter of less than 0.2 m, DNA can be injected into cells for subsequent integration and expression.

The process of micro injection requires careful observation of an embryo to ensure that the inserted DNA is targeted and not injected into other cells in the sample or surrounding tissue. To accomplish this, researchers use an inverted microscope with differential interference contrast (DIC) optics. Micromanipulators are attached to each side of the microscope, one to control the movement of the holding pipette and the other to control the injection needle. A PC with motion control software allows the user to program a sequence of commands for automated manipulation and injection.

In addition to the traditional manual microinjection, a range of automated systems have been developed to improve the efficiency and precision of the process. For example, the MicroJect 1000A can be programmed to deliver a specific volume of reagent or gene directly into an embryo or cell. The system uses a high-pressure, internally-controlled chamber that maintains a stable pressure for a set time period to deliver volumes ranging from femtoliters to microliters.

Besides the automation, a key advantage of the new system is the ability to use a smaller injection volume to reduce the risk of damage to the cell and embryo during the injection process. This is particularly important when working with metaphase-II oocytes, which are more fragile than germinal vesicle stage oocytes and zygotes. The small injection volume allows the researcher to better control the insertion of DNA into the pronucleus of the embryo, and it also increases the likelihood that injected DNA will integrate and express in the host cell.

Prior to injection, single HFF cells obtained by enzymatic dissociation are resuspended in cell culture medium and passed through a 40 mm filter to prevent blocking of the cell holder chip’s designed trapping channels. The cell holder chip is then sterilized and filled with a negative pressure system for cell trapping and connected to the injection device.

The microinjection process begins by locating the pronuclei of the embryo. This can be done by observing the fertilized oocyte through an inverted microscope with DIC optics. The embryo is positioned at the center of the field of view, and the injection needle is placed at the point closest to the pronuclei. The injection needle is then inserted into the pronucleus until the nucleolus becomes visible.

A successful microinjection results in the integration of the DNA into the genome of the host cell. The inserted DNA then undergoes normal gene expression and replication. The DNA can be introduced into the pronucleus of a fertilized oocyte to create a transgenic organism, or into a somatic cell to produce knockout animal models with desirable genetic modifications.

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