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
Isolation of Genetic Material
DNA is extracted from the source (donor) cell.
Restriction Endonucleases (Restriction Enzymes)
Enzymes that cut DNA at specific sequences.
Generate “sticky” or “blunt” ends, which are critical for subsequent DNA recombination steps.
DNA Ligase
Enzyme used to seal nicks and join DNA fragments together (e.g., inserting a gene of interest into a plasmid vector).
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).
Host Organisms
Common hosts include E. coli, yeast (Saccharomyces cerevisiae), and mammalian cell lines.
Chosen based on ease of transformation, expression level, and post-translational modifications needed for the protein.
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.
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.
Screening & Characterization
Colonies are screened for correct DNA insert and protein expression.
Downstream processing includes protein purification, functional assays, and characterization.
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.
