Gene transfer techniques

Gene transfer techniques Success of gene therapy lies in efficient gene transfer into the cell. The gene (cDNA) is generally cloned into a vector to be able to deposit the foreign gene into the target cell. Selection of the right vector is crucial to gene ther-apy. An ideal vector should be able to protect and deliver DNA easily across the cell membrane into the nucleus, should have the ability to regulate expression of the gene of interest and minimize toxicity by targeting gene delivery to specific cells. It should be easy and inexpensive to produce in large quantities. Once the therapeutic gene is cloned into a vector with appropriate regulatory sequences (promoter/enhancer), it is introduced into the target cells. The genes can be delivered either ex vivo –where cells from a selected tissue of the patient are removed, exposed to the gene-transfer vector, selected forthe transgene using markers, and then the genetically cor-rected cells are reintroduced into the patient’s body; or in vivo where the vector DNA is injected directly into the body, generally into the tissue to be treated. Physical and chemical methods of gene transfer Various methods have evolved in the past few years to transfer genes to the target cells. Physical methods such as (a) microinjection of DNA into the cells or (b) electroporation, although very efficient, have their drawbacks in delivering genes in vivo. Also they are expensive as they involve use of specialized instruments. Chemical methods such as (a) calcium phosphate precipitation, where DNA in trapped in a fine precipitate which is endocytosed by the cell, or (b) DNA bound to the positively charged molecules such as DEAE-dextran or polybrene which then bind to the negatively charged cell membrane, are commonly used in the laboratories. DNA encapsulated in synthetic cationic lipid vesicles which fuse with the cell membrane and release DNA into the cell are being used in a number of gene therapy trials 2. Cationic liposome-mediated gene transfer is a safe and effective means of delivering genes directly into tumours. This approach prevents undesirable side effects.

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