What is Genetic Engineering?
- Genetic engineering involves modifying an organism’s genetic material using recombinant DNA (rDNA) technology to introduce desirable traits or produce valuable biological products.
Key Steps in Genetic Engineering
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Isolation of Genetic Material
- DNA is extracted from the source (donor) cell.
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Restriction Endonucleases (Restriction Enzymes)
- Enzymes that cut DNA at specific sequences.
- Generate “sticky” or “blunt” ends, which are critical for subsequent DNA recombination steps.
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DNA Ligase
- Enzyme used to seal nicks and join DNA fragments together (e.g., inserting a gene of interest into a plasmid vector).
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Vectors
- DNA molecules (plasmids, bacteriophages, cosmids) that carry foreign DNA into host cells.
- Key features include an origin of replication (ORI), a selectable marker (e.g., antibiotic resistance), and multiple cloning sites (MCS).
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Host Organisms
- Common hosts include coli, yeast (Saccharomyces cerevisiae), and mammalian cell lines.
- Chosen based on ease of transformation, expression level, and post-translational modifications needed for the protein.
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Transformation and Selection
- Introduction of recombinant DNA into host cells (e.g., via heat shock or electroporation).
- Cells are grown on selective media; only transformants carrying the vector/marker survive or exhibit a detectable trait.
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Expression of the Gene
- Once inside the host, the desired gene is transcribed and translated to produce the target protein.
- Inducible promoters (e.g., lac, T7) can control the timing and level of expression.
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Screening & Characterization
- Colonies are screened for correct DNA insert and protein expression.
- Downstream processing includes protein purification, functional assays, and characterization.
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Applications of Genetic Engineering in Pharmaceuticals
- Production of Recombinant Proteins: Insulin, growth hormone, erythropoietin.
- Gene Therapy: Correcting genetic disorders (e.g., CRISPR-based therapies).
- Vaccine Development: Recombinant hepatitis B vaccine, COVID-19 mRNA vaccines.
- Monoclonal Antibodies: Used for cancer and autoimmune disease treatment.
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