Genetic recombination technology, which introduces a desired gene into a host cell and makes it express the gene, is widely used from insulin production to crop improvement. Let us understand the mechanisms of vectors and transformation.
Basic Knowledge
Vector (carrier): A DNA molecule that carries a foreign gene into a host.
Main vectors:
・Plasmid: A small circular DNA molecule found in bacteria. It can be cut with a restriction enzyme and have a desired gene inserted.
・Viral vector: Retroviruses, adenoviruses, AAV, and others. Used to introduce genes into animal cells.
・YAC/BAC: Yeast/bacterial artificial chromosomes used to clone large DNA fragments.
Steps of genetic recombination:
① Cut the desired gene and vector with the same restriction enzyme → generate complementary sticky ends.
② Use DNA ligase to join the desired gene to the vector (recombinant DNA).
③ Introduce the vector into a host such as Escherichia coli (transformation).
④ Culture the host and mass-produce the desired protein.
Example of application: Insert the human insulin gene into Escherichia coli → produce insulin in large quantities (put into practical medical use in 1982).
Answer: (1) Using the same restriction enzyme produces complementary sticky ends, bringing the desired gene and vector close through hydrogen bonds and making it easier for ligase to join them. (2) DNA ligase reconnects the cleaved phosphodiester bonds through covalent bonds, completing the recombinant DNA molecule. (3) Include a drug-resistance gene, such as an ampicillin-resistance gene, in the vector, and culture the cells in medium containing the drug; only transformants will grow.
- Vector = a DNA molecule that carries a foreign gene into a host
- Plasmid = a small circular bacterial DNA molecule; the most commonly used vector
- Same restriction enzyme → complementary sticky ends → joined by DNA ligase
- Transformation = uptake of foreign DNA resulting in a change in traits
- Selectable marker = a drug-resistance gene used to select transformants
- Recombinant insulin = put into practical use in 1982; revolutionized diabetes treatment
Practice
- Give two reasons why plasmids are used in gene cloning.
- Explain how to identify cells containing the desired gene insert from colonies of transformed Escherichia coli.
- State why viral vectors are more suitable than plasmids for introducing genes into animal cells.