MICROINJECTION GENE TRANSFER METHOD

The direct microinjection of DNA into the cytoplasm or nuclei of cultured cells is sometimes used as a transfection method. It is highly efficient at the level of individual cells. The most significant use of this technique is introduction of DNA into the oocytes, eggs and embryos of animals, either for transient expression analysis (e.g. in fish or Xenopus) or to generate transgenic animals (e.g. mice, Drosophilathis). The procedure is time consuming and only a small number of cells can be treated. Originally, this technique was used for the transformation of cells that were resistant to any other method of transfection. Stable transfection efficiencies are extremely high, in the order of 20%, and very small quantities of DNA are sufficient.
This technique provides direct nuclear delivery of DNA avoiding the endogenous pathway and also ensures that the DNA is delivered intact. Microinjection is suitable for the introduction of large vectors such as YACs into the pronuclei of fertilized mouse eggs. DNA delivered in this manner must be very pure so it needs a lot of preparation as it is necessary to avoid fragmentation. Shearing can also occur in the delivery needle, and large DNA fragments are often protected by suspension in a high salt buffer and/or mixing with polyamines and other protective agents. Now transfection of cultured cells is automated with computer-controlled micromanipulation and microinjection processes as well as the automated production of injection capillaries and the standardization of cell preparation procedure.

1. MICROINJECTION GENE TRANSFER METHOD RANA SAHA B.Pharm . 3 rd Year

2. ABSTRACT The direct microinjection of DNA into the cytoplasm or nuclei of cultured cells is sometimes used as a transfection method. It is highly efficient at the level of individual cells. The most significant use of this technique is introduction of DNA into the oocytes, eggs and embryos of animals, either for transient expression analysis (e.g. in fish or Xenopus) or to generate transgenic animals (e.g. mice, Drosophilathis ). The procedure is time consuming and only a small number of cells can be treated. Originally, this technique was used for the transformation of cells that were resistant to any other method of transfection. Stable transfection efficiencies are extremely high, in the order of 20%, and very small quantities of DNA are sufficient.

3. GENE TRANSFER TECHNIQUES Due to amphipathic nature of the phospholipid bilayer of the plasma membrane, polar molecules such as DNA and protein are unable to freely pass through the membrane. Various physical or mechanical methods are employed to overcome this and aid in gene transfer as listed below- Electroporation Microinjection Particle Bombardment Sonoporation Laser induced Bead transfection

4. MICROINJECTION GENE TRANSFER TECHNIQUE DNA microinjection was first proposed by Dr. Marshall A. Barber in the early of nineteenth century. This method is widely used for gene transfection in mammals. It involves delivery of foreign DNA into a living cell (e.g. a cell, egg, oocyte, embryos of animals) through a fine glass micropipette. The introduced DNA may lead to the over or under expression of certain genes. It is used to identify the characteristic function of dominant genes.

5. PROCEDURE The delivery of foreign DNA is done under a powerful microscope using a glass micropipette tip of 0.5 mm diameter. Cells to be microinjected are placed in a container. A holding pipette is placed in the field of view of the microscope thatsucks and holds a target cell at the tip. The tip of micropipette is injected through the membrane of the cell to deliver the contents of the needle into the cytoplasm and then the empty needle is taken out.

6. FIGURE : Delivery of DNA into a cell through microinjection

7. ADVANTAGES No requirement of a marker gene. Introduction of the target gene directly into a single cell. Easy identification of transformed cells upon injection of dye along with the DNA. No requirement of selection of the transformed cells using antibiotic resistance or herbicide resistance markers. It can be used for creating transgenic organisms, particularly mammals.

8. DISADVANTAGE The procedure is time consuming and Only a small number of cells can be treated.

9. CONCLUSION This technique provides direct nuclear delivery of DNA avoiding the endogenous pathway and also ensures that the DNA is delivered intact. Microinjection is suitable for the introduction of large vectors such as YACs into the pronuclei of fertilized mouse eggs. DNA delivered in this manner must be very pure so it needs a lot of preparation as it is necessary to avoid fragmentation. Shearing can also occur in the delivery needle, and large DNA fragments are often protected by suspension in a high salt buffer and/or mixing with polyamines and other protective agents. Now transfection of cultured cells is automated with computer-controlled micromanipulation and microinjection processes as well as the automated production of injection capillaries and the standardization of cell preparation procedure.

10. REFERENCES National Programme for Technology Enhanced Learning , a project funded by MHRD, Govt. of India. Molecular Biology Of The Gene by James. D. Watson, PEARSON Publication. https://en.wikipedia.org/wiki/Microinjection Biotechnology by P.K.Gupta , Rastogi Publication Biotechnology by B.D.Singh , Kalpana publication

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