鸡胚的壳內电转化

Electroporation uses an electrical current to disrupt the plasma membrane stability, creating temporary pores. This allows DNA to penetrate the membrane. Simultaneously, the electric field causes negatively charged DNA to migrate toward the positive electrode, driving the genetic material into cells on that side of the injection site.

In ovo electroporation allows researchers to deliver genetic material to specific tissues at precise developmental time points within the intact embryo. This localized, stage-specific delivery enables scientists to examine genetic mechanisms governing individual developmental events without disrupting the embryo's natural environment.

Key tools include capillary needles made from pulled glass pipets for injecting DNA, electrodes consisting of exposed wires connected to an electroporator, and a microinjector or mouth pipet for controlling fluid movement. The electroporator generates electrical pulses with adjustable duration, frequency, and voltage, often activated by a foot pedal.

Eggs must be incubated at 37°C until reaching the desired developmental stage. A window is cut in the shell, and physiological salt solution is added to prevent drying. The tissue of interest is positioned under a microscope, and the capillary needle containing the injection solution is carefully pierced into the embryo to deliver the genetic construct.

Electroporation can deliver gene silencing constructs to block expression or plasmids containing protein-encoding genes to promote expression in specific cell populations. Scientists can also introduce DNA constructs encoding fluorescent proteins to visualize cells and structures during development, enabling live imaging of complex developmental processes.

After electroporation, electrodes are removed and wiped with 70% ethanol. Antibiotic-supplemented salt solution is added to prevent infection, and the shell window is sealed with tape. Eggs are returned to the incubator for continued development before phenotypic analysis or imaging to assess the effects of the genetic manipulation.

For embryos older than Hamburger Hamilton stage 20, ex ovo culture provides improved tissue access compared to in ovo procedures. This video focuses on early embryos developing within the shell, but older chicks benefit from external culturing methods that allow better manipulation and visualization of developing tissues.