Guidelines For Marine Protected Areas

Annex: Excerpt from the Guide to the Cartagena Protocol on Biosafety 69

Description of gene constructs used in in vitro nucleic acid techniques

Once a gene has been isolated from a donor organism, it is modified in the laboratory so that it can be inserted effectively into the intended recipient organism. The modifications include making a large number of copies of the gene to be introduced, and possibly introducing changes to the sequence of nucleotides in the isolated gene in specific ways to enhance the expression of the gene once it is introduced into the intended recipient organism.

Following this, the gene to be introduced is built into a “gene construct”. The gene construct includes a “promoter sequence” which is necessary to ensure that the gene is expressed correctly in the recipient organism. Different promoter sequences control gene expression in different ways - some allow continuous expression of the gene, while others switch expression of the gene on or off at different stages of the life-cycle of the organisms, or control the particular tissues or organs in which the gene will be expressed. “Termination”and “signalling” sequences are also incorporated into the gene construct. The termination sequence acts as a signal that flags where the end of the introduced gene is located: like the promoter sequence, the termination sequence is also important in ensuring that the introduced gene is expressed correctly. The signalling sequence provides information about the processing of the product produced from the gene construct.

A “marker” gene is often incorporated into the gene construct - this helps to make it easier to identify which individuals of a recipient organism have been modified by the introduction of the gene construct. Commonly used markers genes are those for antibiotic resistance: following introduction of the gene construct, individuals of the recipient organism are grown in the presence of antibiotics, and under these conditions, only those individuals that have been modified by the gene construct will show antibiotic resistance and therefore will be able to grow. Marker genes may be removed from the LMOs formed by this process at a later stage. Because of concerns over possible spread of antibiotic resistance traits, the use of antibiotic resistance marker genes is being phased out. Finally, a vector may be incorporated into the gene construct. The purpose of the vector is to assist transfer the gene construct into the recipient organism. An example of a gene construct including a bacterial DNA vector (Agrobacterium plasmid), is shown below. The following diagram gives an example of a very simple gene construct: (Note: Gene constructs currently used may include multiple elements - for example, several promoter sequences and desired genes) The gene construct is built from genetic material isolated from several different organisms, for example, a promoter from the Cauliflower Mosaic Virus, a bacterial DNA vector (Agrobacterium plasmid), one or more genes that may have been modified artificially in the laboratory, termination and signalling sequences, and a selectable marker gene, for example for resistance to the antibiotic kanamycin.

69 From Article 3,Use of Terms,paragraph 216,and Box 16,of the Explanatory Guide.

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