Cell Culture and Genetic Manipulation

The One Thing

Cell culture provides controlled conditions for growing and studying cells, while genetic manipulation deliberately changes their DNA to produce or investigate specific traits. Together, they underpin biotechnology applications in medicine, agriculture, industry, and environmental management.

Reactions, Processes and Experiments

Key Terms

• Cell culture: The maintenance and multiplication of cells outside their original organism in a suitable artificial medium.
• Microbial culture: Growth of bacteria, fungi, or other microorganisms in liquid or solid nutrient media.
• Plant tissue culture: The in vitro growth of plant cells, tissues, or organs on a nutrient medium under sterile conditions.
• Animal cell culture: The growth of animal cells in a controlled medium containing nutrients and, often, growth factors.
• In vitro: A process carried out outside the living organism, usually in laboratory glassware or culture vessels.
• Aseptic technique: Methods used to prevent contamination by unwanted microorganisms during culture work.
• Culture medium: A nutrient solution or solid material providing water, salts, carbon sources, nitrogen, vitamins, and other substances needed for cell growth.
• Callus: An unorganized mass of actively dividing plant cells formed from an explant in tissue culture.
• Explant: A piece of plant tissue or organ placed on a culture medium to initiate tissue culture.
• Totipotency: The ability of a plant cell to develop into a complete plant under suitable conditions.
• Micropropagation: Rapid production of genetically similar plants through plant tissue culture.
• Organ culture: Growth of an intact organ or part of an organ while maintaining some of its original structure and function.
• Primary culture: A culture started directly from cells or tissues taken from an organism.
• Cell line: A population of cells maintained through repeated subculturing after being established from a primary culture.
• Subculturing: Transferring cells from an old culture to fresh medium to provide nutrients and space for further growth.
• Suspension culture: A culture in which individual cells or small cell groups grow suspended in a liquid medium.
• Bioreactor: A vessel providing controlled temperature, pH, oxygen, mixing, and nutrient supply for large-scale cell or microbial growth.
• Genetic manipulation: Deliberate alteration, transfer, insertion, deletion, or regulation of genetic material.
• Recombinant DNA: DNA formed by joining genetic material from two or more different sources.
• Restriction enzyme: An enzyme that cuts DNA at specific recognition sequences.
• DNA ligase: An enzyme that joins DNA fragments by forming bonds between adjacent nucleotides.
• Vector: A carrier, such as a plasmid or modified virus, used to transfer a gene into a host cell.
• Plasmid: A small, usually circular DNA molecule in bacteria that can replicate independently and may carry inserted genes.
• Transformation: Introduction of foreign DNA into a cell, especially a bacterial cell.
• Transfection: Introduction of foreign nucleic acids into animal or other eukaryotic cells, commonly by chemical or physical methods.
• Selectable marker: A gene that helps identify cells carrying the desired genetic construct, often by providing resistance to a selective agent.
• Reporter gene: A gene producing an easily detected signal, such as fluorescence or colour, to show whether gene transfer or expression has occurred.
• Gene expression: The process by which information in a gene is used to produce a functional RNA or protein.
• CRISPR-Cas system: A gene-editing method that uses guide RNA and a Cas enzyme to target and modify a chosen DNA sequence.
• Transgenic organism: An organism containing a gene introduced from another organism or species.
• Germplasm conservation: Preservation of genetic material, such as seeds, embryos, cells, or tissues, for future use.

Easily Confused

• In vitro vs in vivo: In vitro processes occur outside the living organism in laboratory vessels; in vivo processes occur inside a living organism.
• Transformation vs transfection: Transformation generally refers to introducing foreign DNA into bacterial cells; transfection refers to introducing foreign nucleic acids into animal or other eukaryotic cells.
• Primary culture vs cell line: A primary culture is started directly from an organism’s cells or tissues; a cell line is maintained through repeated subculturing after establishment.
• Anchorage-dependent vs suspension-growing cells: Anchorage-dependent animal cells require a surface for growth; suspension-growing cells grow while suspended in liquid medium.
• Selectable marker vs reporter gene: A selectable marker identifies cells carrying a construct through survival or resistance under selection; a reporter gene indicates transfer or expression through a detectable signal such as fluorescence or colour.
• Restriction enzyme vs DNA ligase: A restriction enzyme cuts DNA at specific recognition sequences; DNA ligase joins DNA fragments.
• Explant vs callus: An explant is the tissue introduced into culture; callus is the unorganized mass of dividing cells that may form from it.
• Auxin-to-cytokinin ratio vs cytokinin-to-auxin ratio: A higher auxin-to-cytokinin ratio generally favours root formation, whereas a higher cytokinin-to-auxin ratio generally favours shoot formation.
• Solid microbial culture vs liquid microbial culture: Solid media are used for isolated colonies; liquid media are used for biomass and product formation.
• Log phase vs stationary phase: The log or exponential phase involves rapid population increase; the stationary phase occurs when population size levels off.

What Gets Asked

• Describe the stages of a typical culture workflow. Marks depend on including material selection, medium preparation, sterilization, aseptic inoculation, controlled incubation, monitoring, and subculturing where necessary.
• Explain microbial growth using the growth phases and equations. The required relationships are N_t = N_0 x 2^n and g = t/n; confusing exponential growth with stationary or death phase loses marks.
• Explain plant tissue culture and micropropagation. Answers should identify the explant, surface sterilization, callus or shoot initiation, multiplication, rooting, and hardening, rather than describing plant culture only generally.
• Predict the effect of plant growth regulators. A higher auxin-to-cytokinin ratio generally favours roots, while a higher cytokinin-to-auxin ratio generally favours shoots; the response also depends on species and culture conditions.
• Outline recombinant DNA production and identification. The sequence is DNA isolation, restriction-enzyme cutting, DNA-ligase joining, introduction into a host cell, selection, and expression or product recovery; selectable markers and reporter genes have distinct roles.
• Explain PCR, gel electrophoresis, or CRISPR-Cas. PCR requires denaturation, primer annealing, and extension; in gel electrophoresis smaller DNA fragments move faster toward the positive electrode; CRISPR-Cas uses guide RNA and a Cas enzyme to target DNA.